The Wuerzburg hybridoma library against Drosophila brain.

Abstract

This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

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The Wuerzburg Hybridoma Library against Drosophila BrainThe Wuerzburg Hybridoma Library against Drosophila Brain
Alois Hofbauer ab; Thomas Ebel b; Bernhard Waltenspiel a; Peter Oswald b; Yi-chun Chen b; Partho Halder b;
Saskia Biskup b; Urs Lewandrowski c; Christiane Winkler c; Albert Sickmann c; Sigrid Buchner b; Erich Buchner
b
a Institut für Zoologie, Lehrstuhl für Entwicklungsbiologie, Regensburg, Germany b Theodor-Boveri-Institut für
Biowissenschaften, Lehrstuhl für Genetik und Neurobiologie, Würzburg, Germany c Rudolf-Virchow-Center for
Experimental Biomedicine, Wuerzburg, Germany
First Published on: 08 January 2009
To cite this Article To cite this Article Hofbauer, Alois, Ebel, Thomas, Waltenspiel, Bernhard, Oswald, Peter, Chen, Yi-chun, Halder, Partho, Biskup,
Saskia, Lewandrowski, Urs, Winkler, Christiane, Sickmann, Albert, Buchner, Sigrid and Buchner, Erich(2009)'The Wuerzburg
Hybridoma Library against Drosophila Brain',Journal of Neurogenetics,
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The Wuerzburg Hybridoma Library against Drosophila Brain
Alois Hofbauer,1,2Thomas Ebel,2Bernhard Waltenspiel,1Peter Oswald,2Yi-chun Chen,2
Partho Halder,2Saskia Biskup,2Urs Lewandrowski,3Christiane Winkler,3Albert Sickmann,3
Sigrid Buchner2and Erich Buchner2
1Institut fu ¨r Zoologie, Lehrstuhl fu ¨r Entwicklungsbiologie, Regensburg, Germany
2Theodor-Boveri-Institut fu ¨r Biowissenschaften, Lehrstuhl fu ¨r Genetik und Neurobiologie, Wu ¨rzburg, Germany
3Rudolf-Virchow-Center for Experimental Biomedicine, Wuerzburg, Germany
Abstract: This review describes the present state of a project to identify and characterize novel nervous system proteins by using
monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of
homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by
these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the
approach ‘‘from antibody to gene’’, several genes coding for novel proteins of the presynaptic terminal were cloned and characterized.
These include the ‘‘cysteine string protein’’ gene (Csp, mAb ab49), the ‘‘synapse-associated protein of 47 kDa’’ gene (Sap47, mAbs
nc46 and nb200), and the ‘‘Bruchpilot’’ gene (brp, mAb nc82). By a ‘‘candidate’’ approach, mAb nb33 was shown to recognize the
pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and
calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against
mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of ‘‘epidermal growth factor receptor
pathway substrate clone 15’’ (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we
review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of
mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of
identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.
Keywords: brain proteins, CSP, synapsin, SAP47, Bruchpilot, calbindin, PDF, Eps15
INTRODUCTION
The brain is distinguished from other organs by its
enormous complexity, both at the structural and the
molecular levels. In order to understand the development
of neuronal networks and how the various cell types
operate and interact to initiate, maintain, modulate, and
terminate behavior, it is necessary to identify and localize
the proteins that are specifically expressed in the brain as
a prerequisite for the study of their molecular, cellular,
and systemic function. Of particular interest are those
proteins that are cell-specifically expressed and are
responsible for the differentiation of neurons and glial
cells into thousands of different cell types with their
individual molecular, structural, and functional character-
istics. One approach to the identification and localization
of brain proteins makes use of the exquisite ability of the
mammalian adaptive immune system to recognize foreign
proteins, which can be exploited by the generation of
monoclonal antibodies (mAbs) against brain homoge-
nates. This approach has been used successfully with
various organisms, and in Drosophila, it has been
pioneered many years ago by the group of the late
Seymour Benzer (Fujita et al., 1982; Fujita, 1988). The
present review summarizes the information on the genes
and proteins identified by Abs of the Wuerzburg
hybridoma library (Hofbauer, 1991) (Table 1). In this
project, we intended to find Abs that bind to specific cells
or compartments of the Drosophila brain and use them to
identify the genes coding for the corresponding antigens.
MATERIALS AND METHODS
Fly Strains
Wild-type Berlin and Canton S were used as wild-type
control and for the staining on cryostat sections. The
Received 7 August 2008; Revised 5 September 2008; Accepted 9 September 2008
Address correspondence to Alois Hofbauer, Institut fu ¨r Zoologie, Lehrstuhl fu ¨r Entwicklungsbiologie, Universita ¨tsstr. 31, D-93047
Regensburg, Germany. E-mail: alois.hofbauer@biologie.uni-regensburg.de
J. Neurogenetics, 2009: 1?14, iFirst article
Copyright # 2008 Informa UK Ltd.
ISSN: 0167-7063 print/1563-5260 online
DOI: 10.1080/01677060802471627
1
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Pdf01mutant (Renn et al., 1999), as well as lines with a
modified expression of the Pdf gene, were obtaind from
Paul Taghert: y w;; P{w?mF6-2} Pdf01expresses AA 1-
82 of the pigment dispersing factor (PDF) precursor with
the PDF peptide replaced by Locusta myosupressin. In y
w; P{w?mE10-2A}; Pdf01PDF is expressed, whereas
the rest of the precursor is replaced by preproFMRFamide
sequences.
Immunizations
Inbred BALBc mice (Charles River WIGA, Sulzfeld,
Germany) were used for immunizations throughout. The
injections were done mostly intraperitoneally, sometimes
in combination with subcutaneous injections (aa, ab, and
ca). Adjuvans was used only when purified proteins were
injected. Homogenates were prepared by using a glass
homogenator (Dounce, 2 mL). We tried different im-
munization strategies by using either brains or heads,
combinations of mutants and wild type, and immuniza-
tions of newborns with mutants missing parts of the visual
system in combination with later immunizations, using
wild type, to provoke Abs against the differences between
mutant and wild-type heads. There might have been some
effects, but we could not find any mutant-specific
differences; therefore, no further details are presented
here. mAbs Pok13 and 3C11 were generated by injecting
calbindin32 and synapsin, respectively, that had been
cleaved from bacterially expressed glutathione S-transfer-
ase (GST) fusion protein purified by glutathion-agarose
from bacterial lysate. The general pattern of immuniza-
tions is presented in Table 2.
Cell Culture
Hybridoma medium consisted of 0.7 RPMI 1640
(7 parts), 0.2 medium 199 (2 parts), and 0.1 fetal calf
serum (FCS) (1 part) and was hybridoma tested (Invitro-
gen, Paisley, UK). Selection medium (HAT medium)
consisted of hybridoma medium with a hypoxanthin-
aminopterin-thymidin cocktail added. Polyethylene glycol
(PEG) solution consisted of PEG 4000 (Merck, Darm-
stadt, Germany), 1 g/mL in RPMI, plus 10% (vol)
dimethyl sulfoxide (DMSO).
The fusion procedure was based on the methods
described in Zola and Brooks (1982). Immunized mice
were sacrificed, then the spleen was removed and
mazerated between the frosted ends of sterile glass slides.
To the cell suspension, about 2 x 107myeloma cells were
added. The cells were washed in RPMI medium and
centrifuged. The pellet was slowly and gently resus-
pended by adding 1 mL of PEG solution over 1 minute.
After another 90 seconds, the suspension was gradually
diluted by adding 10 mL of RPMI in increasing amounts
over the next 2 minutes. After pelleting the cells, the
supernatant was replaced by cell-culture medium and after
3 minutes by pure hybridoma tested FCS. The resus-
pended cells were individually seeded in cell-culture
plates (96 wells) containing equilibrated HAT medium.
Cells were grown to cultures, and aliquots were frozen as
well as samples of supernatants. Cell lines producing Abs
as confirmed by enzyme-linked immunosorbent assay
(ELISA) were subcloned. Subclones were accepted as the
origin for cell lines if microscopic examination showed
only one clone per well.
ELISA
To test cell lines for the production of Abs, we used the
TSP system (Nunc, Langenselbold, Germany). With this
technique, Abs were adsorbed to plastic knobs inserted
into the culture wells. The Abs were then detected by
using the ABC technique (Vector, Burlingame, Califor-
nia, USA) with horseradish peroxidase (HRP) as enzyme
and catechol/phenylendiamine as chromogen (Hanker
et al., 1977).
Immunohistochemistry
Phosphate-buffered paraformaldehyde (4%) was used for
fixation. Flies were cold anesthetized, in ice-cold fixative
the proboscis and the air sacks of the head were removed,
and the flies were fixed for 2 hours. The fixative was
replaced by 25% sucrose overnight. Heads were em-
bedded in methyl cellulose and frozen rapidly in liquid
nitrogen. Cryostat sections (10 or 20 mm) were used for
immunohistochemical staining. The hybridoma super-
natant was used at a dilution of 1?1 in phosphate-
buffered saline (PBS) for the first try and adjusted to
optimal dilution in later experiments. The primary Ab was
detected by using the ABC elite kit (Vector Laboratories,
Burlingame, CA, USA) with HRP as the detecting
enzyme and diaminobenzidine (Sigma-Aldrich, Tauf-
kirchen, Germany) as chromogen. All reaction solutions
contained 0.1% TritonX100.
Mass Spectrometry (MS)
Subsequent to gel electrophoretic separations, gel spots/
bands of interest were excised and proteins reduced,
carbamidomethylated, and tryptically digested, as de-
scribed previously (Schindler et al., 2008). The resulting
peptide mixtures were subjected to nanoscale liquid
chromatographic separation coupled online to a LTQ-XL
mass spectrometer (Thermo-Fischer Scientific, Dreieich,
2A. Hofbauer et al.
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Page 4

Germany). An UltiMate 3000 high-performance liquid
chromatography (HPLC) system (Dionex, Idstein, Germany)
was used in combination with a preconcentration setup
comprising a 100-mm inner diameter?2 cm length
precolumn and a 75-mm inner diameter?15 cm length
separation column, both with Synergy Hydro-RP material
(Phenomenex, Aschaffenburg, Germany). Peptides were
separated by using a binary gradient system (solvent A:
0.1% formic acid in water; solvent B: 84% acetonitrile,
0.1% formic acid). Online mass spectrometric detection by
LTQ-XL was achieved by using a duty cycle consisting of
a single survey scan, followed by five tandem-MS scans of
selected precursors.
Mass spectrometric data interpretation was performed
by using the Mascot Algorithm (version 2.1; Matrix
Science, London, UK) and Mascot Daemon (version
2.1.6). Searches were conducted against Flybase (http://
flybase.bio.indiana.edu/) with the following settings.
Carbamidomethylation (C) was set as fixed and oxidation
(M) as variable modification. Trypsin, with one misclea-
vage site, was chosen as protease, while precursor and
fragment-ion tolerance was set to 1.5 Da. Identified
peptides were validated manually after identification by
the algorithm.
RESULTS
The number of genes expressed in the developing, or the
adult, nervous system is of the order of several thousands,
and probably more than half of them are still unknown.
We, therefore, injected heterogeneous cocktails of possi-
ble antigens obtained by homogenizing native or chemi-
cally fixed isolated brains or whole heads into mice and
fused their spleen cells with myeloma cells to obtain
about 1,000 hybridoma cell lines. The supernatants of
these were screened for immunoglobulin content by
ELISA and for staining of brain structures by immuno-
histochemistry on frozen head sections. This approach
resulted in a collection of about 200 mAbs directed
against diverse antigens of the Drosophila brain. This
library was supplemented by a few hybridoma clones,
which were obtained after immunization of mice with
affinity-purified tagged proteins of bacterially expressed
Drosophila cDNAs, which had been isolated from a
cDNA expression library due to cross-reactions with
antisera against mammalian proteins.
Histological Staining Patterns
After immunizations with a complex mixture of antigens,
as contained in the crude homogenates of brains or heads,
one has to expect Abs directed against a wide variety of
different types of antigens. Because we characterized the
Abs in a first screen according to their staining pattern on
cryostat sections after formaldehyde fixation, the general
patterns we describe here refer to anatomical and
histological aspects of the nervous system. They are not
exclusive, as some Abs show overlapping patterns.
A minority of Abs label different body tissues or
cellular compartments, such as the trachea, cell-body
layers due to specific nuclear staining, muscle, cuticula, or
the perineureum (Figure 1).
In the retina, several Abs recognize specific cell
types, such as receptor cells or pigment cell types,
staining whole cells or cell compartments. Some are
A
B
C
D
Figure 1. Tissue-specific staining patterns in the Drosophila
brain. (A) Antibody nc7 is specific for chitin and, therefore,
demonstrates the tracheal system in the brain. (B) Selective
staining of all cell nuclei (ga21). (C) nc120 binds exclusively to
the perineureal sheeth. (D) ab49 binds to cysteine string protein
and stains all neuropil. Horizontal cryostat sections, scale bar:
50 mm.
Monoclonal Antibodies for Drosophila Nervous System Analysis3
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restricted to the retina, whereas some also stain other
areas of the brain (Figure 2). One of these Abs, nb181, is
specific to all photoreceptor cells staining their full cell
morphology. It was used to describe the eyelet, a group of
four extraretinal photoreceptor cells at the posterior rim of
the compound eye (Hofbauer and Buchner, 1989). These
receptor cells originate from the Bolwig organ, the larval
photoreceptor organ (Helfrich-Fo ¨rster et al., 2002).
Recently, it was shown that during metamorphosis, the
surviving photoreceptor cells of the eyelet switch from
rhodopsin-5 to -6 gene expression (Sprecher and Desplan,
2008).
One class of Abs appeared to stain many cells, but
differentiated between various components of the cells.
Most conspicuous among these are Abs selective for
synaptic neuropil regions (Figure 3A?3H). Some of them
do not bind to perikarya, but stain neuropil regions rather
uniformly, presumably representing the density of sy-
napses because the antigen is contained in all synapses,
while others clearly differentiate between different neu-
ropil areas. This was most obvious in the neuropil areas of
the optic lobes, there revealing characteristic patterns of
layers mainly in the medulla (Figure 3). One of these Abs,
nb236, shows staining exclusively in the lamina neuropil
(Figure 3F). It might be specific for a type of lamina glia.
The largest class of Abs, also the class most difficult
to characterize, comprises Abs that clearly bind to a few
individual elements in the nervous system. Typically,
these Abs stain a small number of cell bodies and only
parts of their arborizations or even only substructures of
their terminals (Figure 4A?4E). Again, this can be
demonstrated most easily in the optic lobes, where
many Abs reveal individual cell bodies in the area
between the anterior medulla neuropil and the central
brain, as well as a pattern of dots in one or few distinct
layers of the medulla neuropil, possibly representing
terminal boutons of tangential cells (Figure 4D 4E). We
found no morphologically distinct staining of repetitive
cells in the optic lobes, such as constituents of medulla
columns or lamina cartridges.
Abs staining small cell populations in the optic lobes
usually also stained cells in the central brain. These cell
bodies were scattered in the cell-body layers, most
frequently in the posterior cortex and in the dorsal area
in or near the pars intercerebralis (Figure 4A and 4C).
Terminal staining was most conspicuous in the central
ABCD
F
E
Figure 2. Different staining patterns in the Drosophila retina indicating selective staining of cell types or cell compartments. (A)
antibody nc133, (B) ab160, (C) ab79, and (D) nc42. (E) Antibody nc10 is selective for retinal and antennal cells innervating a
subpopulation of antennal glomeruli. (F) ab47 stains tangential cells in the medulla and further cells in the central brain, in addition to
strong retina staining. AG, antennal glomeruli; M, medulla; R, retina. Horizontal cryostat sections, scale bars: 50 mm.
4 A. Hofbauer et al.
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complex, especially the fan-shaped body, but was found
also in other areas (Figure 4C?4E).
Only in a small number of cases did the Ab bind to
all parts of a cell, dendrites, axons, neurite, and cell body,
such that the complete morphology of the targeted cells
was revealed. An obvious example for such a fortunate
case is nb33, an Ab that selectively stains cells expressing
the pigment dispersing factor gene (Pdf) (Figure 4B).
Antigen Analysis
We have used several of our Abs to identify the
corresponding antigen and its encoding gene. Three
approaches have been used: 1) screening a cDNA
expression library with the antibody and verification of
positive clones by generating new mouse polyclonal
antisera against the bacterially expressed cDNA-encoded
protein; whenever the Western signal and the staining
pattern on brain sections of the mAb matched with the
signals obtained by the new polyclonal antisera, one could
be reasonably certain that the correct gene was identified;
2) a second approach uses the mAb to affinity purify the
antigen from head homogenates by immunoprecipitation.
Alternatively, in favorable cases, the antigen can be
purified by two-dimensional (2-D) gel electrophoresis.
The purified protein can then be subjected to MS analysis
and identified by comparison of the observed peptide
fingerprint with the Drosophila proteome inferred from
the genome sequence; and 3) the third*candidate*
approach can be applied only if the cells that are stained
by the mAb are known such that one can speculate which
proteins might specifically be expressed in these cells.
Final proof for the correct association of mAb and gene is
obtained in all three situations when the staining is
eliminated in a null mutant for the identified gene.
Table 1 lists the mAbs of the Wuerzburg hybridoma
library, for which the antigen has been identified or for
which some information has been published. The present
knowledge about the identified proteins is briefly re-
viewed in the following paragraphs.
mAb ab49: Cysteine String Protein (CSP)
The mAb, ab49, was selected for the first large screens of
a cDNA expression library that had been generated by
K.E.Z. in our group. mAb ab49 stains all neuropil (Figure
1D) and synaptic boutons on adult and larval muscles. A
number of clones from the same gene were isolated with
mAb ab49. To verify that these clones indeed code for the
ab49 antigen, bacterially expressed GST fusion protein
was purified and injected into mice whose antisera
showed a staining pattern essentially identical to mAb
ab49. By dot blots of sequential decapeptides, it was later
shown that mAb ab49 recognizes the amino-acid epitope,
FTGA (Arnold et al., 2004). Cysteine string proteins are
conserved cysteine-rich molecular cochaperones for
Hsp70 family chaperones and are attached to vesicles of
synapses and secretory organs by a string of palmitoylated
cysteine residues (Buchner and Gundersen, 1997; Cham-
berlain and Burgoyne, 2000; Zinsmaier and Bronk, 2001;
Evans et al., 2003). Deletion of the Csp gene in
Drosophila impairs synaptic transmission, especially at
high temperatures (Zinsmaier et al., 1994; Umbach et al.,
1994; Eberle et al., 1998; Bronk et al., 2005). CSP is also
required for nerve terminal growth and prevention of
neurodegeneration in Drosophila and mice (Ferna ´ndez-
Chaco ´n et al., 2004; Chandra et al., 2005; Schmitz et al.,
2006; Dawson-Scully et al., 2007). Transgenic pan-neural
expression of wild-type cDNAs in the Csp null mutants
rescues the temperature-sensitive paralytic phenotype.
However, if cDNA constructs lacking the codons for the
cysteine string are used, targeting of CSP to vesicular
membranes fails and no phenotypic rescue is observed
(Arnold et al., 2004). A similar defect of CSP targeting is
observed when palmitoylation of the cysteine string is
prevented by mutation of the palmitoyl transferase
A
C
E
GH
F
D
B
Figure 3. Staining patterns of neuropil structures, as demon-
strated in the optic lobes. Selective specificities to cell types or
subcellular structures result in characteristic layering patterns.
(A) ca51, (B) ab49, (C) nb43, (D) na21, (E) nc82, (F) nb236, (G)
aa2, and (H) nc46. CB, Cucatti’s bundle of the medulla; iC, inner
chiasm; La, lamina; Lo, lobula; Lp, lobula plate; M, medulla; R,
retina. Horizontal cryostat sections, scale bars in E for A?E, in H
for F?H: 50 mm.
Monoclonal Antibodies for Drosophila Nervous System Analysis5
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huntingtin-interacting protein 14 (HIP14) (Ohyama et al.,
2007; Stowers and Isacoff, 2007). In vertebrates, CSPs
have been shown to form a trimeric complex with HSC70
(70-kDa heat-shock cognate protein) and SGT (small
glutamine-rich tetratricopeptide repeat domain protein)
that can function as a guanine nucleotide exchange factor
(GEF) for Gas (Swayne et al., 2006). Various interaction
partners of CSPs have been described, such as G-proteins,
cystic fibrosis transmembrane conductance regulators, a-
synuclein, huntingtin, HIP14, and myosin 1e, but the
molecular function of CSP that prevents the temperature-
sensitive failure of synaptic exocytosis observed in
Drosophila Csp null mutants remains to be elucidated.
mAbs nc46 and nb200: Synapse-associated
Protein of 47 kDa (SAP47)
These two antibodies stained all neuropil similar to Figure
1D. By screening the Zinsmaier cDNA expression library
with MAB nc46, five independent cDNAs were isolated,
but protein expressed from only one clone generated
antisera with a similar staining pattern as nc46. This clone
identified the Sap47 gene of Drosophila (Reichmuth et
al., 1995) and was shown to recognize an epitope in the
N-terminal decapeptide, FSGLTNQFTS. A second Ab,
mAb nb200, showed very similar staining, both on
sections and in Western blots, and later was shown to
recognize the same protein (epitope QQAKHF in a central
domain). Very little is known about the protein encoded
by the Sap47 gene. Homologs can be found in sea
anemones, sea urchins, insects, frogs, fish, and mammals,
but no information is available on their function. The
encoded polypeptides belong to a novel superfamily of
proteins containing the ‘‘BSD’’ domain, which is found
in conserved transcription factors (e.g., BTF2 of mam-
mals), synapse-associated proteins (e.g., SAP47 of Dro-
sophila), and DOS2-like proteins (e.g., DOS2 of yeast)
(Doerks et al., 2002). Drosophila Sap47 null mutants are
viable and fertile (Funk et al., 2004) but show complex
behavioral phenotypes, which are presently investigated.
SAP47 largely colocalizes with synapsins to synaptic
terminals but is also found in axons and in some perikarya
(Reichmuth et al., 1995). Interestingly, preliminary
experiments indicate an interaction between Drosophila
SAP47 and synapsin. Intrigued by the fact that mAb nc46
also stains synaptic regions in fish and mouse brain
(Reichmuth et al., 1995), we used immunoprecipitation
and MS (Protana, Denmark) to identify the proteins
recognized in vertebrate brains. It turned out that mAb
nc46, which in Drosophila specifically detects the
synapse-associated protein, SAP47, does not recognize
the mouse homolog of SAP47, but cross-reacts with
mouse synapsin I and immunoglobulin heavy-chain
binding protein (BiP). In mouse-brain sections, nc46
shows staining similar to Abs against the synaptic protein,
synaptophysin (Figure 5). The epitope recognized by
mAb nc46 in Drosophila SAP47 (FSGLTNQFT) is not
found in the mammalian synapsin I or the BiP sequence.
Thus, the cause of this cross-reaction remains unknown.
Table 1. List of antibodies which have been published or for which the antigen is known
AntibodyStains FigureAntigen (approach) Reference
aa2
ab43
ab47
ab49
ca8
ca51
fb45
nc82
All neuropil
Few cells
Few cells
All neuropil
Few cells
Antennae, layers in visual system 3A
Mushroom bodies, visual system
All neuropil, presynaptic active
zone
PDF-neurons
Photoreceptors
Antennae, layers in visual system
Antennae, layers in visual system 3D
Retina, antennae, olfactory glo-
meruli
nc46 nb200 All neuropil
3GEPS15 (2)
Buchner et al. (1988)
Buchner et al. (1988)
Buchner et al. (1988), Zinsmaier et al. (1990)
Buchner et al. (1988)
Buchner et al. (1988), Sto ¨rtkuhl et al. (1994)
Bicker et al (1993)
Wagh et al. (2006), Kittel et al. (2006)
4D, E
1D, 3B CSP (1)
3EBruchpilot (2)
nb33
nb181
nb230
na21
nc10
4B PDF precursor (3)
Hofbauer and Buchner, 1989
Sto ¨rtkuhl et al. (1994)
Sto ¨rtkuhl et al. (1994)
Sto ¨rtkuhl et al. (1994)2E
5, 3H (mouse synapsin),
SAP47 (1)
calbindin-32 (1)
synapsin (1)
Reichmuth et al. (1995), Funk et al. (2004)
pok13
3C11
Numerous cells, selected neuropil 6
All neuropil
Reifegerste et al. (1993)
Klagges et al. (1996), Godenschwege et al. (2004)
Approaches: 1, screen of cDNA expression libraries; 2, protein isolation and mass spectrometry; 3, candidate approach.
6 A. Hofbauer et al.
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mAb 3C11 (anti-SYNORF1): Synapsin
The Synapsin gene of Drosophila was cloned by chance
due to the cross-reaction of an unrelated peptide antiserum
at a time when unsuccessful homology screens with
vertebrate synapsin cDNA sequences had led to the
speculation that invertebrates might not have synapsin
homologs. The isolated cDNA clone was tagged and
expressed in Escherichia Coli, and the purified protein
was injected in mice for the generation of mAbs. The
clone, 3C11, produced the strongest staining of most or
all synaptic terminals (similar to Figure 3H) and was
selected for further analysis. The recognized epitope,
LFGGMEVCGL, lies in the conserved C domain, such
that mAb 3C11 binds to synapsins of many invertebrate
species. The synapsin gene (Syn) has several unusual
features. On its opposite strand lies the Timp gene
overlapping with intron 9 and exons 8 and 9 of Syn.
This nested organization of Syn and Timp genes has been
conserved in evolution (Pohar et al., 1999; Yu et al.,
2003). Translation of the Syn mRNA apparently starts at a
noncanonical CTG codon, as no ATG is found between an
upstream in-frame stop codon and the codons for the
conserved A-domain (Klagges et al., 1996). This con-
jecture is supported by N-terminal peptide sequencing by
Edman degradation (Godenschwege et al., 2004). Another
striking feature is the TAG stop codon that separates two
large open-reading frames and is found in all known
cDNAs of Syn. Translation termination at this stop codon
leads to isoforms of 70?80 kDa, while isoform(s) of about
140 kDa are generated presumably by TAG read-through
(Godenschwege et al., 2004). Alternative splicing, as a
mechanism to remove the stop codon, seems less likely,
because transgenic expression of the largest cDNA in Syn
null mutants produces both the large and the small
isoforms, and no high-score splice sites are detected in
the cDNA sequence that could explain the removal of the
stop codon as part of an alternatively spliced intron. Yet
another intriguing feature of the Drosophila synapsin gene
is the fact that its pre-mRNA is very efficiently edited by
the enzyme, adenosine deaminase acting on RNA
(ADAR) (Diegelmann et al., 2006). The encoding of the
consensus sequence, RRXS, for phosphorylation by
protein kinase A (PKA) and calcium/calmodulin depen-
dent kinases I and IV (CamK-I and -IV) in the conserved
‘‘A’’ domain of vertebrate synapsins is conserved in the
genomic DNA of Drosophila but modified to RGXS in
most or all mature transcripts of late larvae, pupae, and
adults. The N-terminal decapeptide, containing the edited
sequence, is not significantly phosphorylated by bovine
PKA in vitro, while the genomically encoded decapeptide
is an excellent substrate (Diegelmann et al., 2006). The in-
vivo consequences of Syn mRNA editing are presently
investigated.
Vertebrate synapsins are believed to regulate the
trafficking of synaptic vesicles between reserve and
cycling pools, but also may have other roles, as they
show structural similarity to ATPases and interact with
sarcoma (Src) kinase to enhance their activity in synaptic
vesicle fractions (Hilfiker et al., 1999, 2005; Baldelli
A
B
C
D
E
Figure 4. Selective staining of small neuronal subpopulations in the Drosophila brain. (A) A cluster of neurons stained in the pars
intercerebralis (ab135). (B) nb33 is specific for Pdf-expressing neurons. Here, one cell body and arborizations in the most distal medulla
neuropil are shown. (C?E) Scattered cell bodies and traces of arborizations in the central brain. (C) nc53, (D), and (E) ab47 (two sections
out of a series). AG, antennal glomeruli; CC, central complex; P, peduncule of the mushroom bodies. Horizontal cryostat sections, scale
bars (in C for C?E): 50 mm.
Monoclonal Antibodies for Drosophila Nervous System Analysis7
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Page 9

et al., 2005; Onofri et al., 2007). They are substrates for
various kinases, including PKA and CamK-I, which
phosphorylate the conserved N-terminal ‘‘A’’ domain.
Extensive attempts have been made to elucidate the
cellular and systemic function of Drosophila synapsins.
Null mutants with large or small deletions in the Syn gene
are viable and fertile but show altered responses in
various behavioral paradigms, including several learning
assays (Godenschwege et al., 2004; Michels et al., 2005).
Electron microscopy and basic electrophysiology of larval
neuromuscular junctions failed to reveal clear differences
between wild-type and Syn null mutants, but recent FM1-
43 staining demonstrated altered distributions of recycling
and reserve pools of synaptic vesicles in type-Ib boutons
of the Syn97null mutant (Akbergenova & Bykhovskaia,
2007). The role that the editing of synapsin pre-mRNA
plays for learning and memory and interaction with
SAP47 are major unsolved problems.
mAb nc82: Bruchpilot Protein (BRP)
During a sabbatical of SB and EB in the laboratory of
Veronica Rodrigues in 2000?2001, it was noted that mAb
nc82, which in adults, stains all neuropil (Figure 3E),
binds in larval nerve-muscle preparations selectively to
small spots within each synaptic bouton. The obvious
speculation that these spots might represent active zones
was first verified by Wucherpfennig et al. (2003) by
demonstrating a close association of nc82 staining with
the distribution of postsynaptic glutamate receptors. By
2D-electrophoresis and Western blotting, two protein
spots could be isolated and analyzed by MALDI-TOF,
which identified a protein encoded by the annotated gene,
CG30337. Reverse-transcriptase polymerase chain reac-
tion (RT-PCR) experiments and sequencing in collabora-
tion with the Sigrist group connected the three annotated
‘‘genes,’’ CG12933, CG30336, and CG30337, to a single
large gene, which received the name ‘‘bruchpilot’’ (brp)
(German for crash pilot), based on the unstable flight of
animals panneurally expressing a brp-RNAi construct.
The largest known BRP isoform of 190 kDa contains
domains with high homology to the mammalian active
zone protein, ELKS/CAST/ERC, and a large C-terminal
coiled-coil domain. Other isoforms are yet to be char-
acterized. The brp knock-down animals also showed
almost no spontaneous walking activity, and in larval
neuromuscular preparations, the quantal content of ex-
citatory junction currents (EJCs) was reduced to about
50%. At the ultrastructural level, presynaptic electron
dense projections (T-bars) were absent from adult photo-
receptor terminals and drastically reduced in larval
neuromuscular boutons (Wagh et al., 2006). In sub-
diffractionresolutionSTED
depletion), fluorescence microscopy BRP is arranged in
donut-shaped rings at the center of active zones. Null
mutants are lethal, but synaptic boutons of larval escapers
can be investigated. They entirely lack T-bars, display
presynaptic membrane ‘‘rufflings,’’ show increased min-
iature EJC amplitude, a 70% reduction in EJC quantal
content, and transient short-term facilitation. Also, the
expression of GFP-labeled presynaptic voltage-sensitive
calcium channels was severely reduced at larval synaptic
boutons lacking BRP, suggesting that in wild-type
synapses, BRP is involved in concentrating calcium
channels near docking sites for synaptic vesicles (Kittel
et al., 2006a). Obvious questions on the function of BRP,
(stimulated emission
A
B
C
D
E
G
H
F
Figure 5. Immunohistochemical localization of the monoclonal
antibody (mAb) nc46 antigen in mouse brain. (A) negative
control without first antibody. (B) Positive control with a
polyclonal antibody against GFAP, a marker of astrocytes,
dilution 1:1,000. (C, E, and G) Section stained with mAb nc46,
dilution 1:100, at 10, 20, and 40 times magnification. (D) Section
treated with a polyclonal antiserum against the synaptic protein
synaptophysin, dilution 1:2,000. CA, cornu ammonis; mo,
stratum moleculare; gr, stratum granulare; py, stratum pyrami-
dale; luc, stratum lucidum.
8A. Hofbauer et al.
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Page 10

such as its interactions with other proteins and its role
during active zone assembly, remain to be solved (Kittel
et al., 2006b).
mAb aa2: Epidermal Growth Factor Receptor
Pathway Substrate Clone 15 (EPS-15)
This Ab (Figure 3G) recognized a protein present in all
synapses, both in adults and in larvae. Confocal double-
labeling experiments, during a sabbatical of EB and SB in
the laboratory of K. Zinsmaier on larval neuromuscular
boutons with mAb aa2 and rabbit antidynamin serum
(kindly provided by Mani Ramaswami), indicated colo-
calization of the two antigens. Initial attempts to purify
the aa2 antigen by immunoprecipitation failed, but after
raising the hybridoma cells in serum-free medium (to
eliminate bovine immunoglobulins, which compete with
aa2 for binding to the protein-G agarose), two gel pieces,
containing a 100-kDa Coomassie-stained band, were
subjected to analysis by a linear ion-trap mass spectro-
meter (LTQ LC/MS). Among 25 proteins of the Droso-
phila proteome with peptide patterns similar to proteins in
the gel pieces (scores above 150), four were known
components of the presynaptic endocytosis complex of
Drosophila (dynamin, EPS-15, a-adaptin, dynamin-asso-
ciated protein 160; DAP160). Since mAb aa2 did not
recognize GFP-labeled dynamin (flies kindly provided by
Richard Ordway), we tested wild-type and homozygous
EPS-15 null mutant larvae (escapers) with mAb aa2 and
anti-EPS-15 antiserum (mutants and antiserum kindly
provided by Hugo Bellen) and found that aa2, like anti-
EPS-15 antiserum, failed to label synaptic terminals of the
mutant. We, thus, conclude that mAb aa2 most likely
binds to EPS-15.
mAb na21
The staining of this Ab in the antennae of Drosophila has
briefly been described previously (Sto ¨rtkuhl et al., 1994).
Here, we include the staining pattern of na21 in the adult
optic lobes (Figure 3D), which closely resembles that of
an Ab against choline acetyl transferase (Chat) (Buchner
et al., 1986). The identification of the na21 antigen is
presently attempted.
mAb pok13: Calbindin
Vertebrate parvalbumin, calbindin D-28k, and calretinin
are calcium-binding proteins with four to six EF-hand
calcium-binding domains. In the brain, they are expressed
at high levels in various cell types; parvalbumin is often
colocalized with the neurotransmitter, gamma aminobu-
tyric acid (GABA). These proteins presumably function
both as calcium buffers and calcium sensors and are
important for calcium homeostasis. Human calbindin
could play a role in nitric oxide regulation, as 80% of
its cysteines can be S-nitrosylated (Tao et al., 2002). It has
been shown that calbindin interacts with caspase-3 and
protects the cells against apoptosis (Bellido et al., 2000).
Knock-out mice lacking calretinin and/or calbindin suffer
from ataxic movement disorders, which are accompanied
by fast network oscillations in the cerebellar cortex
(Cheron et al., 2004), while parvalbumin deficiency leads
to a propensity to epileptic seizures (Schwaller et al.,
2004). In flies, only a single gene codes for a protein that
shows high homology to these related calcium-binding
proteins. It has been identified by screening the Zinsmaier
l-gt11 head cDNA expression library with an antiserum
against carp parvalbumin. It codes for six EF-hand
domains and has been named the calbindin-32 gene
(Cbn) due to the apparent size of the encoded protein
(Reifegerste et al., 1993) (synonyms: calbindin 53E;
Cbp53E). Spleen cells of a mouse injected with purified
cDNA-encoded protein were fused with myeloma cells,
the hybridoma cells were screened to isolate the mAb,
pok13, which binds to calbindin both on Western blots
and in histochemical preparations (Figure 6). mAb pok13
stains certain muscles and a large number of neurons,
such as in the distal lamina (probably monopolar cells
L2), a group of cell bodies caudal to the medulla
(probably T2 and/or T3) (Fischbach and Dittrich, 1989),
and cells in the third antennal segment. While most
neuropil stains weakly, several regions of the central-
brain neuropil are conspicuously devoid of staining, such
as the ellipsoid body, the fan-shaped body, and the
mushroom bodies. Attempts to isolate mutants or P-
insertions in the Cbn gene have failed so far, and apart
from its calcium-binding property, as indicated by a shift
in apparent molecular weight in the presence of calcium
(Figure 6C), at present no functional information on
calbindin in Drosophila is available.
mAb nb33: Pigment Dispersing Factor (PDF)
Precursor
nb33 stains, very selectively, both the ‘‘large ventral
lateral neurons’’ and the ‘‘small ventral lateral neurons’’
in Drosophila (lLNv, sLNv), in addition to a few cells in
the abdominal ganglia. The lateral neurons are known to
be clock neurons containing the peptide PDF, the
Drosophila homolog of the crustacean peptide pigment
dispersing hormone (PDH) (Helfrich-Fo ¨rster & Homberg,
1993). Indeed, the nb33 staining pattern is essentially
identical to the pattern observed with an antiserum against
crab PDH, and the antibody has been used as an
alternative to anti-PDH serum (Veleri et al., 2003;
Monoclonal Antibodies for Drosophila Nervous System Analysis 9
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Page 11

Helfrich-Fo ¨rster et al., 2007). There might be subtle
differences in the circadian distribution of the antigen and
in the staining intensity of cell compartments, but the
morphology of stained cells is identical.
The Drosophila Pdf gene codes for a putative 102-
amino-acid precursor protein; only the last 18 amino acids
represent the final PDF peptide. This peptide shows high
homology to the PDH of crustaceans and to the PDF of
other Drosophilids and other insects. In contrast, the
remaining precursor sequence appears much more vari-
able (Park & Hall, 1998). In experiments on Pdf01, a null
mutant for PDF, we obtained no staining with nb33 nor
with anti-PDH. Transgenic flies expressing a modified
PDF precursor without the final PDF sequence on a Pdf01
background showed normal nb33 staining, but no staining
with anti-PDH serum. Flies expressing a precursor
truncated to the final PDF sequence produced no staining
with nb33, but we obtained normal staining with the
serum against PDH (fly strains kindly provided by P.
Taghert). Thus, nb33 recognizes an epitope in the
precursor region, which is not contained in the final
PDF peptide.
We compared the nb33 staining pattern of D.
melanogaster with several other Drosophila species.
There is virtually no difference in the staining among
the species of the melanogaster species subgroup tested
(D. melanogaster, D. teissieri, D. yakuba, D. simulans).
Of the other species subgroups combined in the melano-
gaster species group, we found weak staining in D.
eugracilis, D. ananassae, and D. saltans. The staining in
this species was less distinct, often reduced to the staining
of cell bodies only. In addition, cross-reactions of the Ab
with antigens in additional cell nuclei and cell-body layers
became evident. No staining was obtained in D. pseu-
doobscura. (subgenus Sophophora) and D. virilis (sub-
genus Drosophila).
Considering that the epitope of nb33 is somewhere
on the precursor outside the PDF peptide domain, it is not
surprising that the species range of nb33 is much more
restricted, compared to the anti-PDH serum (Na ¨ssel et al.,
1993, Helfrich-Fo ¨rster et al., 1998).
DISCUSSION
This review summarizes the information available for
several novel brain proteins identified by the approach
from antibody to gene and, for the first time, presents the
immunohistochemical staining patterns for a number of
unpublished Abs from the Wuerzburg hybridoma library
in a generally accessible form. In the original screen of the
library, these staining patterns served as the selection
criterion. By this procedure, we primarily obtained a
collection of Abs that reveal anatomical and morpholo-
gical details of the Drosophila brain. A subgroup of Abs
was then used to identify the corresponding antigens and
their encoding genes. Even without any knowledge about
the antigens or genes, the library of Abs is useful for
descriptive neuroanatomical work. Details revealed by
A
B
C
D
Figure 6. Staining patterns of pok13 directed against Droso-
phila calbindin. (A) In the dorsal protocerebrum, numerous cell
bodies and the ‘‘unstructured’’ neuropil are labeled, while the
mushroom bodies, the fan-shaped body, and the ellipsoid body
show very low staining. (B) The horizontal section at the level of
the esophagus canal illustrates the distribution of CBN immu-
nopositive cell bodies, specifically in the lamina cortex and
posterior to the medulla (possibly of L2 and T2/T3 neurons,
respectively). (C) Cells in the third antennal segment are labeled.
Inset: Coomassie staining of 7.5% SDS gel loaded with affinity-
purified bacterially expressed Drosophila calbindin (lanes 1 and
2) or the GST-tag Sj-26 (lanes 3 and 4) in the presence of 1 mM
CaCl2 (lanes 1 and 3) or 5 mM EGTA (lanes 2 and 4). (D) Direct
flight muscles (dFMs) show strong staining, while indirect flight
muscles (iFMs) and tergotrochanter muscle (TTM) lack CBN.
Ant, antennae; Re, retina; La, lamina; Me, medulla; Lo, lobula;
LP, lobula plate; Pk, mushroom body; Ek, ellipsoid body; Fk,
fan-shaped body.
10A. Hofbauer et al.
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such Abs can be compared between different species or
between wild-type und morphological mutants affecting
the nervous system. In addition, one of the synapse-
specific antibodies, nc82, is widely used for convenient
staining of all neuropil to obtain a framework for cell-
specific labeling by other Abs or green flourescent protein
(GFP) expression. This Ab was actually used for estab-
lishing a standard reference tool for neuroanatomical
work on Drosophila (Rein et al., 2002). Since nc82
exclusively binds to a small spot in each synapse (the
active zone), the brain remains transparent in confocal
immunofluorescence microscopy of whole-mount pre-
parations.
The route from a mAb to the identification of its
antigen and the corresponding gene is not necessarily
straightforward. Screening of cDNA expression libraries
with the mAb cannot be successful if the epitope
recognized by the Ab is generated by post-translational
modification. Also, conformational epitopes that require
chaperones for correct folding may not form in the
bacterial environment of the expression clones. Another
problem of most cDNA expression libraries is the fact that
the cDNAs are inserted into the phage with random
orientation and random reading frame. Thus, only one of
six clones can, in principle, contain the correct coding
sequence, and five of six clones may generate new
epitopes that are not present in the fly but might cross-
react with the Ab and produce false-positive signals. It is,
therefore, not surprising that in our screens, we always
had false-positive clones necessitating considerable effort
to identify the correct clone. Oligo-dT primed cDNA
libraries, in addition, are likely to contain many 5’
incomplete cDNAs and thus may lack epitopes near the
N-termini of the encoded proteins. Attempts with this
approach to identify the antigens of various mAbs that
only bind to few brain cells, including mAbs nb33, ca8,
nb236, and fa56, produced only false-positive clones,
indicating that in addition to the mentioned limitations,
this approach can be expected to be successful only if the
mRNA encoding the antigen is relatively abundant in the
head poly-A?mRNA fraction.
MS of trypsin-digested proteins, and comparison of
the observed peptide spectra with the predicted peptides
of all hypothetical proteins encoded by the whole genome
of the species under study, has become a powerful
technique for the identification of isolated proteins. For
Drosophila, this approach became feasible in 2000, when
the nonrepetitive genome sequence became available
(Adams et al., 2000).
From Table 1, it is apparent that it has been possible
to identify the antigens for several mAbs. However, both
molecular approaches worked, so far, only for antigens,
which are present in most or all neurons. The only antigen
that is expressed exclusively in a small number of neurons
was identified by the candidate approach, making use of
knowledge on cells with a similar gestalt in other species.
Apparently, the number of cDNA copies in head libraries
and the amount of antigen in head homogenates are too
low for a successful molecular approach. Thus, it seems
necessary to enrich mRNAs or proteins only from cells
positive for the mAb. This could, perhaps, be achieved by
dissociating the cells from immature brains and subjecting
them to fluorescence-activated cell sorting.
Alternative approaches to identify cell-specifically
expressed genes have been developed in recent decades.
Embryonic expression patterns were determined for 44%
of the protein-coding genes of Drosophila in a large-scale
approach using in-situ hybridization and quantitative
microarray time-course analysis (Tomancak et al.,
2007). For adult flies, the enhancer trap system (O’Kane
& Gehring, 1987; Brand & Perrimon, 1993) has received
the highest attention. The most extensive collection of
some 4,000 enhancer trap lines has been generated by a
Japanese consortium headed by K. Ito (available from the
Bloomington stock center). However, many enhancer trap
lines suffer from limited specificity of the expression
drivers, since not all regulatory elements of a gene can be
expected to interact with the promotor used in the
enhancer trap construct in exactly the same fashion as
with its endogenous promotor. In addition, the expression
patterns may depend on the genomic insertion site of the
transposon relative to the promotor, and even variability
Table 2. Series of immunizations
CodeAntigen Immunization newborns Immunization (juveniles, adults)
aa ab
ca
fa fb
ga
na nb nc
po
3C
Fixed brains, freeze-dried
Brains, freeze-dried
Heads
Heads
Heads
Dmel\calbindin-32
Dmel\synapsin
?
sol (?s)
mnb (?s)
rol sol (?s)
mnb sol
?
?
Wild-type
sol (?s) wild-type
mnb (?s) wild-type
rol sol (?s) wild-type
mnb sol (?s) wild-type
Purified protein (?F)
Purified protein (?F)
Code refers to the first two characters of the name of the hybridoma clone, with the first letter referring to the immunization regime,
the second letter distinguishing individual animals. ?s, immunization followed by immunsuppression (cycloheximide); F, Freund’s
adjuvant.
Monoclonal Antibodies for Drosophila Nervous System Analysis 11
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of expression, depending on the UAS-reporter used, has
been described for various Gal4 lines (Ito et al., 2003; A.
Jenett, personal communication). Thus, it is not surprising
that the cell-specific all-or-none staining observed with
the majority of our mAbs is only rarely seen in enhancer-
trap-reporter stainings. Attempts to overcome these
limitations of the enhancer trap system use the ‘‘gene
trap’’ (or fly trap) technique which requires a P
transposon construct containing an artificial exon coding
for the reporter to insert into an intron, thus generating a
fusion protein with the endogenous expression pattern
(Kelso et al., 2004; Buszczak et al., 2007). The important
advantage of these techniques is the possibility of a
straightforward identification of the gene coding for a
protein of the observed expression pattern.
CONCLUSION
In conclusion, we predict that the mAbs of our library,
which selectively stain only a few neurons, will be useful
for reproducible display of cell morphology, but that the
systematic identification of the protein recognized by the
Ab will require new, sophisticated approaches.
ACKNOWLEDGMENTS
The authors thank Dagmar Richter and Dieter Dudaczek
for their excellent technical assistance, Hugo Bellen and
Karen L. Schulze for providing Eps15 mutants, antiserum,
and advice, Heiner Dircksen for providing the anti-PDH
antibody, Paul Taghert, Susan Renn, and Mei Han for
providing fly strains, and the DFG for financial support to
E.B. (SFB581/B6,B21, HO798/5, and GK1156) and to
A.H. (SFB798/5).
REFERENCES
Adams, M. D. et al. (2000). The genome sequence of
Drosophila melanogaster. Science, 287, 2185?2195.
Akbergenova, Y., & Bykhovskaia, M. (2007). Synapsin main-
tains the reserve vesicle pool and spatial segregation of the
recycling pool in Drosophila presynaptic boutons. Brain
Res, 1178, 52?64.
Arnold, C., Reisch, N., Leibold, C., Becker, S., Pru ¨fert, K.,
Sautter, K., et al. (2004). Structure-function analysis of the
cysteine string protein in Drosophila: cysteine string,
linker, and C-terminus. J Exp Biol, 207, 1323?1334.
Baldelli, P., Fassio, A., Corradi, A., Cremona, O., Valtorta, F., &
Benfenati, F. (2005). Synapsins and neuroexocytosis:
recent views from functional studies on synapsin null
mutant mice. Arch Ital Biol, 143, 113?126.
Bellido, T., Huening, M., Raval-Pandya, M., Manolagas, S. C.,
& Christakos S. (2000). Calbindin-D28k is expressed in
osteoblastic cells and suppresses their apoptosis by inhibit-
ing caspase-3 activity. J Biol Chem, 275, 26328?26332.
Bicker, G., Kreissl, S., & Hofbauer, A. (1993). Monoclonal
antibody labels olfactory and visual pathways in Droso-
phila and Apis brains. J Comp Neurol, 335, 413?424.
Brand, A. H., & Perrimon, N. (1993). Targeted gene expression
as a means of altering cell fates and generating dominant
phenotypes. Development. 118, 401?415.
Bronk, P., Nie, Z., Klose, M. K., Dawson-Scully, K., Zhang, J.,
Robertson, R. M., et al. (2005). The multiple functions of
cysteine-string protein analyzed at Drosophila nerve term-
inals. J Neurosci, 25, 2204?2214.
Buchner, E., Bader, R., Buchner, S., Cox, J., Emson, P. C.,
Flory, E., et al. (1988). Cell-specific immuno-probes for the
brain of normal and mutant Drosophila melanogaster. I.
Wild-type visual system. Cell Tissue Res, 253, 357?370.
Buchner, E., Buchner, S., Crawford, G., Mason, W. T.,
Salvaterra, P. M., & Sattelle, D. B. (1986). Choline
acetyltransferase-like immunoreactivity in the brain of
Drosophila melanogaster. Cell Tissue Res, 246, 57?62.
Buchner, E., & Gundersen, C. B. (1997). The DnaJ-like cysteine
string protein and exocytotic neurotransmitter release.
Trends Neurosci, 20, 223?227.
Buszczak, M., Paterno, S., Lighthouse, D., Bachman, J., Planck,
J., Owen, S., et al. (2007). The carnegie protein trap library:
a versatile tool for Drosophila developmental studies.
Genetics, 175, 1505?1531.
Chamberlain, L. H., & Burgoyne, R. D. (2000). Cysteine-string
protein: the chaperone at the synapse. J Neurochem, 74,
1781?1789.
Chandra, S., Gallardo, G., Ferna ´ndez-Chaco ´n, R., Schlu ¨ter, O.
M., & Su ¨dhof, T. C. (2005). Alpha-synuclein cooperates
with CSPalpha in preventing neurodegeneration. Cell, 123,
383?396.
Cheron, G., Gall, D., Servais, L., Dan, B., Maex, R., &
Schiffmann, S. N. (2004). Inactivation of calcium-binding
protein genes induces 160-Hz oscillations in the cerebellar
cortex of alert mice. J Neurosci, 24, 434?441.
Cheron, G., Servais L., & Dan, B. (2004). Cerebellar network
plasticity: from genes to fast oscillation. Neuroscience, 153,
1?19.
Dawson-Scully, K., Lin, Y., Imad, M., Zhang, J., Marin, L.,
Horne, J. A., et al. (2007). Morphological and functional
effects of altered cysteine string protein at the Drosophila
larval neuromuscular junction. Synapse, 61, 1?16.
Diegelmann, S., Nieratschker, V., Werner, U., Hoppe, J., Zars,
T., & Buchner, E. (2006). The conserved protein kinase-A
target motif in synapsin of Drosophila is effectively
modified by pre-mRNA editing. BMC Neurosci, 7, 76.
Eberle, K. K,, Zinsmaier, K. E., Buchner, S., Gruhn, M., Jenni,
M., Arnold, C., et al. (1998). Wide distribution of the
cysteine string proteins in Drosophila tissues revealed by
targeted mutagenesis. Cell Tissue Res, 294, 203?217.
Doerks, T., Huber, S., Buchner, E., & Bork, P. (2002). BSD: a
novel domain in transcription factors and synapse-asso-
ciated proteins. Trends Biochem Sci, 27, 168?170.
12 A. Hofbauer et al.
Downloaded At: 16:13 29 January 2009

Page 14

Evans, G. J., Morgan, A., & Burgoyne, R. D. (2003). Tying
everything together: the multiple roles of cysteine string
protein (CSP) in regulated exocytosis. Traffic, 4, 653?659.
Ferna ´ndez-Chaco ´n, R., Wo ¨lfel, M., Nishimune, H., Tabares, L.,
Schmitz, F., Castellano-Mun ˜oz, M., et al..(2004). The
synaptic vesicle protein CSP alpha prevents presynaptic
degeneration. Neuron, 42, 237?251.
Fischbach, K.-F., & Dittrich, A. P. M. (1989). The optic lobe of
Drosophila melanogaster. I. Golgi analysis of wild-type
structure. Cell Tissue Res, 258, 441?475.
Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A. Shotwell, S.
(1982). Monoclonal antibodies against the Drosophila
nervous system. Proc Natl Acad Sci U S A, 79, 7929?7933.
Fujita, S. C. (1988). Use of hybridoma libraries in the study of
the genetics and development of Drosophila. Ann Rev
Entomol, 33, 1?15.
Funk, N., Becker, S., Huber, S., Brunner, M., & Buchner, E.
(2004). Targeted mutagenesis of the Sap47 gene of
Drosophila: Flies lacking the synapse associated protein
of 47 kDa are viable and fertile. BMC Neuroscience, 5, 16.
Godenschwege, T. A., Reisch, D., Diegelmann, S., Eberle, K.,
Funk, N., Heisenberg, M., et al. (2004). Flies lacking all
synapsins are unexpectedly healthy but are impaired in
complex behaviour. Europ J Neurosci, 20, 611?622.
Hanker, J. S., Yates, P. E., Metz, C. B., & Rustioni, A. (1977). A
new, specific, sensitive, and noncarcinogenic reagent for
the demonstration of horseradish peroxidase. Histochem J,
9, 789?792.
Helfrich-Fo ¨rster, C., & Homberg, U. (1993). Pigment-dispersing
hormone-immunoreactive neurons in the nervous system of
wild-type Drosophila melanogaster and of several mutants
with altered circadian rhythmicity. J Comp Neurol, 337,
177?190.
Helfrich-Fo ¨rster, C., Stengl, M., & Homberg, U. (1998).
Organization of the circadian system in insects. Chronobiol
Internat, 15, 567?594.
Helfrich-Fo ¨rster, C., Edwards, T., Yasuyama, K., Wisotzki, B.,
Schneuwly, S., Stanewsky, R., et al. (2002). The extra-
retinal eyelet of Drosophila: development, ultrastructure,
and putative circadian function. J Neurosci, 22, 9255?9266.
Helfrich-Fo ¨rster, C., Shafer, O. T., Wu ¨lbeck, C., Grieshaber, E.,
Rieger, D., & Taghert, P. (2007). Development and
morphology of the clock-gene-expressing lateral neurons
of Drosophila melanogaster. J Comp Neurol, 500, 47?70.
Hilfiker, S., Pieribone, V. A., Czernik, A. J., Kao, H. T.,
Augustine, G. J., & Greengard, P. (1999). Synapsins as
regulators of neurotransmitter release. Philos Trans R Soc
Lond B Biol Sci, 354, 269?279.
Hilfiker, S., Benfenati, F., Doussau, F., Nairn, A. C., Czernik, A.
J., Augustine, G. J., et al. (2005). Structural domains
involved in the regulation of transmitter release by
synapsins. J Neurosci, 25, 2658?2669.
Hofbauer, A. (1991). (A library of monoclonal antibodies
against the brain of Drosophila melanogaster). Professorial
dissertation, University of Wu ¨rzburg, Germany.
Hofbauer, A., & Buchner, E. (1989). Does Drosophila have
seven eyes? Naturwiss, 76, 335?336.
Ito, K., Okada, R., Tanaka, N. K., & Awasaki, T. (2003).
Cautionary observations on preparing and interpreting brain
images using molecular biology-based staining techniques.
Microsc Res Tech, 62, 170?186.
Kelso, R. J., Buszczak, M., Quin ˜ones, A. T., Castiblanco, C.,
Mazzalupo, S., & Cooley, L. (2004). Flytrap, a database
documenting a GFP protein-trap insertion screen in
Drosophila melanogaster. Nucleic Acids Res, 32, D418?
D420.
Kittel, R. J., Wichmann, C., Rasse, T. M., Fouquet, W.,
Schmidt, M., Schmid, A., et al. (2006a). Bruchpilot
promotes active zone assembly, Ca2?channel clustering,
and vesicle release. Science, 312, 1051?1054.
Kittel, R. J., Hallermann, S., Thomsen, S., Wichmann, C.,
Sigrist, S. J., & Heckmann, M. (2006b). Active zone
assembly and synaptic release. Biochem Soc Trans, 34,
939?941.
Klagges, B. R. E., Heimbeck, G., Godenschwege, T., Hofbauer,
A., Pflugfelder, G. O., Reifegerste, R., et al. (1996).
Invertebrate synapsins: a single gene codes for several
isoforms in Drosophila. J Neurosci, 16, 3154?3165.
Michels, B., Diegelmann, S., Tanimoto, H., Schwenkert, I.,
Buchner E., & Gerber B. (2005). A role for synapsin in
associative learning: the Drosophila larva as a study case.
Learn Mem, 12, 224?231.
Na ¨ssel, D. R., Shiga, S., Mohrherr, C. J., & Rao, K. R. (1993).
Pigment-dispersing hormone-like peptide in the nervous
system of the flies Phormia and Drosophila: immunocy-
tochemistry and partial characterization. J Comp Neurol,
331, 183?198.
Ohyama, T., Verstreken, P., Ly, C. V., Rosenmund, T., Rajan,
A., Tien, A. C., et al. (2007). Huntingtin-interacting protein
14, a palmitoyl transferase required for exocytosis and
targeting of CSP to synaptic vesicles. J Cell Biol, 179,
1481?1496.
O’Kane, C. J., & Gehring, W. J. (1987). Detection in situ of
genomic regulatory elements in Drosophila. Proc Natl
Acad Sci U S A, 84, 9123?9127.
Onofri, F., Messa, M., Matafora, V., Bonanno, G., Corradi, A.,
Bachi, A., et al. (2007). Synapsin phosphorylation by SRC
tyrosine kinase enhances SRC activity in synaptic vesicles.
J Biol Chem, 282, 15754?15767.
Park, J. H., & Hall, J. C. (1998). Isolation and chronobiological
analysis of a neuropeptide pigment-dispersing factor gene
in Drosophila melanogaster. J Biol Rhythms, 13, 219?228.
Pohar, N., Godenschwege, T. A., & Buchner, E. (1999).
Invertebrate tissue inhibitor of metalloproteinase: structure
and nested gene organization within synapsin locus is
conserved from Drosophila to man. Genomics, 57, 293?
296.
Reichmuth, C., Becker, S., Benz, M., Debel, K., Reisch, D.,
Heimbeck, G., et al. (1995). The sap47 gene of Drosophila
melanogaster codes for a novel conserved neuronal protein
associated with synaptic terminals. Mol Brain Res, 32, 45?
54.
Reifegerste, R., Grimm, S., Albert, S., Lipski, N., Heimbeck, G.,
Hofbauer, A., et al. (1993). An invertebrate calcium-
binding protein of the calbindin subfamily: protein struc-
ture, genomic organization, and expression pattern of the
calbindin-32 gene of Drosophila. J Neurosci, 13, 2186?
2198.
Monoclonal Antibodies for Drosophila Nervous System Analysis 13
Downloaded At: 16:13 29 January 2009

Page 15

Rein, K., Zo ¨ckler, M., Mader M. T., Gru ¨bel, C., & Heisenberg,
M. (2002). The Drosophila standard brain. Curr Biol, 12,
227?231.
Renn, S. C., Park, J. H., Rosbash, M., Hall, J. C., & Taghert, P.
H. (1999). A pdf neuropeptide gene mutation and ablation
of PDF neurons each cause severe abnormalities of
behavioral circadian rhythms in Drosophila. Cell, 99,
791?802.
Schindler, J., Lewandrowski, U., Sickmann, A., & Friauf, E.
(2008). Aqueous polymer two-phase systems for the
proteomic analysis of plasma membranes from minute
brain samples. J. Proteome Res., 7, 432?442.
Schmitz, F., Tabares, L., Khimich, D., Strenzke, N., de la Villa-
Polo, P., Castellano-Mun ˜oz, M., et al. (2006). CSPalpha-
deficiency causes massive and rapid photoreceptor degen-
eration. Proc Natl Acad Sci U S A, 103, 2926?2931.
Schwaller, B., Tetko, I. V., Tandon, P., Silveira, D. C.,
Vreugdenhil, M., Henzi, T., et al. (2004). Parvalbumin
deficiency affects network properties resulting in increased
susceptibility to epileptic seizures. Mol Cell Neurosci, 25,
650?663.
Sprecher, S. G., & Desplan, C. (2008). Switch of rhodopsin
expression in terminally differentiated Drosophila sensory
neurons. Nature, 454, 533?537.
Sto ¨rtkuhl, K. F., Hofbauer, A., Keller, V., Gendre, N., &
Stocker, R. F. (1994). Analysis of immunocytochemical
staining patterns in the antennal system of Drosophila
melanogaster. Cell Tissue Res, 275, 27?38.
Stowers, R. S., & Isacoff, E. Y. (2007). Drosophila huntingtin-
interacting protein 14 is a presynaptic protein required for
photoreceptor synaptic transmission and expression of the
palmitoylated proteins synaptosome-associated protein 25
and cysteine string protein. J Neurosci, 27, 12874?12883.
Swayne, L. A., Beck, K. E., & Braun J. E. (2006). The cysteine
string protein multimeric complex. Biochem Biophys Res
Commun, 348, 83?91.
Tao, L., Murphy, M. E., & English, A. M. (2002). S-nitrosation
of Ca(2?)-loaded and Ca(2?)-free recombinant calbindin
D(28K) from human brain. Biochemistry, 41, 6185?6192.
Tomancak, P., Berman, B. P., Beaton, A., Weiszmann, R.,
Kwan, E., Hartenstein, V., et al. (2007). Global analysis of
patterns of gene expression during Drosophila embryogen-
esis. Genome Biol, 8, R145.
Umbach, J. A., Zinsmaier, K. E., Eberle, K. K., Buchner, E.,
Benzer, S., & Gundersen, C. B. (1994). Presynaptic
dysfunction in Drosophila csp mutants. Neuron, 13, 899?
907.
Veleri, S., Brandes, C., Helfrich-Fo ¨rster, C., Hall, J. C., &
Stanewsky, R. (2003). A self-sustaining, light-entrainable
circadian oscillator in the Drosophila brain. Curr Biol, 13,
1758?1767.
Wagh, D. A., Rasse, T. M., Asan, E., Hofbauer, A., Schwenkert,
I., Du ¨rrbeck, H., et al. (2006). Bruchpilot, a protein with
homology to ELKS/CAST, is required for structural
integrity and function of synaptic active zones in Droso-
phila. Neuron, 49, 833?844.
Wucherpfennig, T., Wilsch-Bra ¨uninger, M., & Gonza ´lez-Gaita ´n,
M. (2003). Role of Drosophila Rab5 during endosomal
trafficking at the synapse and evoked neurotransmitter
release. J Cell Biol, 161, 609?624.
Yu, W. P., Brenner, S., & Venkatesh, B. (2003). Duplication,
degeneration, and subfunctionalization of the nested sy-
napsin-Timp genes in Fugu. Trends Genet, 19, 180?183.
Zinsmaier, K. E., & Bronk, P. (2001). Molecular chaperones and
the regulation of neurotransmitter exocytosis. Biochem
Pharmacol, 62, 1?11.
Zinsmaier, K. E., Hofbauer, A., Heimbeck, G., Pflugfelder, G.
O., Buchner, S., & Bucher, E. (1990). A cysteine-string
protein is expressed in retina and brain of Drosophila.
J Neurogen, 7, 15?29.
Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N., &
Benzer, S. (1994). Paralysis and early death in cysteine-
string protein mutants of Drosophila. Science, 263, 977?
980.
Zola, H., & Brooks, D. (1982) Techniques for the production
and characterisation of monoclonal hybridoma antibodies.
In: Hurrell, J. G. R. (Ed.), Monoclonal Hybridoma
Antibodies: Techniques and Applications (pp. 1?57).
Boca Raton, Florida, USA: CRC Press.
14 A. Hofbauer et al.
Downloaded At: 16:13 29 January 2009