Pinocchio, a novel protein expressed in the antenna, contributes to olfactory behavior inDrosophila melanogaster

Pinocchio, a Novel Protein Expressed in the Antenna,Contributes to Olfactory Behaviorin Drosophila melanogaster

Stephanie M. Rollmann,1,3 Trudy F. C. Mackay,2,3 Robert R. H. Anholt1,2,3

1 Department of Zoology, North Carolina State University, Campus Box 7617, Raleigh,North Carolina 27695-7617

2 Department of Genetics, North Carolina State University, Campus Box 7617, Raleigh,North Carolina 27695-7617

3 W. M. Keck Center for Behavioral Biology, North Carolina State University,Campus Box 7617, Raleigh, North Carolina 27695-7617

Received 1 September 2004; accepted 3 October 2004

ABSTRACT: Most organisms depend on chemore-

ception for survival and reproduction. In Drosophila mel-anogaster multigene families of chemosensory receptors

and putative odorant binding proteins have been identi-

fied. Here, we introduce an additional distinct protein,

encoded by the CG4710 gene, that contributes to olfac-

tory behavior. Previously, we identified through P[lArB]-

element mutagenesis a smell impaired (smi) mutant,

smi21F, with odorant-specific defects in avoidance

responses. Here, we show that the smi21F mutant also

exhibits reduced attractant responses to some, but not all,

of a select group of odorants. Furthermore, electroanten-

nogram amplitudes are increased in smi21F flies. Charac-

terization of flanking sequences of the P[lArB] insertionsite, complementation mapping, phenotypic reversion

through P-element excision, and expression analysis

implicate a predicted gene, CG4710, as the candidate smi

gene. CG4710 produces two transcripts that encode pro-

teins that contain conserved cysteines and which are

reduced in the smi21F mutant. Furthermore, in situ

hybridization reveals CG4710 expression in the third

antennal segment. We have named this gene of

previously unknown function and its product ‘‘Pinocchio

(Pino)’’. ' 2005 Wiley Periodicals, Inc. J Neurobiol 63: 146–158, 2005

Keywords: olfaction; P-element mutagenesis; behavioral

genetics; chemoreception

INTRODUCTION

Chemoreception is the principle modality by which

many organisms gain information from their external

environment and is critical for an organism’s survival

and for modulating social interactions. In recent years

significant progress has been made in our understand-

ing of the molecular mechanisms underlying odor

recognition, but the link between odor perception and

odor-guided behavior remains poorly understood.

With its sequenced genome and well developed

genetic technology, Drosophila melanogaster pro-

vides a powerful model system to investigate how

ensembles of genes mediate odor-guided behavior.

Insect olfactory systems are remarkably similar in

functional organization to those of vertebrates in that

peripheral chemosensory neurons converge on a few

output neurons in distinct spherical structures of neu-

ropil called ‘‘glomeruli’’ (reviewed by Hildebrand

Correspondence to: R. R. H. Anholt ([email protected]).Contract grant sponsor: National Institutes of Health; contract

grant numbers: GM059469, GM45344, and GM45146.Contract grant sponsor: W. M. Keck Foundation.

# 2005 Wiley Periodicals, Inc.Published online 17 February 2005 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/neu.20123

146

and Shepherd, 1997). Insect olfactory systems, how-

ever, are quantitatively less complex than their

vertebrate counterparts. The olfactory system of

D. melanogaster contains only about 1200 chemosen-

sory neurons located in the third antennal segments

and the maxillary palps (reviewed by Stocker, 1994).

Chemosensory neurons from the antennae and maxil-

lary palps project to 43 identified glomeruli in each

antennal lobe (Laissue et al., 1999; Vosshall et al.,

2000; Gao et al., 2000), a small number compared to

the 1800 glomeruli in the mouse olfactory bulb

(Pomeroy et al., 1990).

D. melanogaster was the first insect species in

which families of olfactory receptors (Clyne et al.,

1999; Vosshall et al., 1999; Gao and Chess, 1999)

and gustatory receptors (Scott et al., 2001; Dunipace

et al., 2001) were identified. The genome of D. mela-nogaster contains approximately 60 putative olfac-

tory receptors (Rubin, 2000), similar to the number of

glomeruli in each antennal lobe (Laissue et al., 1999).

Odorant receptors have distinct expression patterns in

the antennae and/or maxillary palps, although one

receptor, Or83b, is expressed ubiquitously (Vosshall

et al., 1999, 2000) and is essential for transporting

other odorant receptors to the dendritic membranes

(Larsson et al., 2004). Electrophysiological studies

demonstrated that olfactory neurons in both the max-

illary palps and the third antennal segments are com-

partmentalized according to strict pairing rules so

that the same pairs of neurons with distinct odorant

specificities are always housed together in the same

sensilla (de Bruyne et al., 1999, 2001). Olfactory neu-

rons expressing the same odorant receptor project

bilaterally to one, or sometimes two, glomeruli in

each antennal lobe (Vosshall et al., 2000; Gao et al.,

2000), displaying a similar convergence to that

observed in the vertebrate olfactory system (Ressler

et al., 1994; Vassar et al., 1994; Mombaerts et al.,

1996). Systematic single cell tracing experiments

have characterized individual projection neurons

from defined glomeruli in the antennal lobe to higher

brain regions, the mushroom body and lateral horn of

the protocerebrum (Wong et al., 2002; Marin et al.,

2002). Elegant electrophysiological studies, in which

odorant receptors were misexpressed in sensilla con-

taining neurons that lacked their endogenous odorant

receptor, have identified the response spectra of a

wide array of Drosophila odorant receptors (Hallem

et al., 2004).

In addition to families of chemosensory receptors,

a large family of odorant binding proteins (OBPs) is

also differentially expressed (Hekmat-Scafe et al.,

1997, 2002; Park et al., 2000; Galindo and Smith,

2001; Shanbhag et al., 2001; Vogt et al., 2002). OBPs

are secreted into the sensillar perilymph and,

although their precise function has not been conclu-

sively established, a prevailing hypothesis is that they

facilitate transport of hydrophobic odorants through

the perilymph to receptors at the chemosensory mem-

brane (Kaissling, 1996; Du and Prestwich, 1995;

Robertson et al., 1999; Vogt et al., 1999). OBPs form

a structurally diverse family of proteins characterized

by six cysteines at conserved positions (Vogt et al.,

1991). Crystallographic studies on the pheromone

binding protein of the silk moth Bombyx mori suggestthat the cysteines stabilize a binding pocket that

undergoes pH-dependent conformational changes for

the binding and release of the pheromone, bombykol

(Leal et al., 1999; Wojtasek and Leal, 1999; Sandler

et al., 2000; Damberger et al., 2000; Leal, 2000;

Horst et al., 2001). In Drosophila, an OBP null

mutant, lush, showed impaired avoidance responses

to ethanol and propanol (Kim et al., 1998; Kim and

Smith, 2001), and the crystal structure of the alcohol

binding site of the Lush protein has been described

(Kruse et al., 2003). The functional relationships

between OBPs and chemosensory receptors and phys-

iological responsiveness of identified olfactory neu-

rons in the antennae and the maxillary palps,

however, remain to be clarified. Here, we introduce

an additional protein, encoded by the CG4710 gene,

which is distinct from the classic OBP family, but

also contributes to olfactory behavior.

MATERIALS AND METHODS

Fly Stocks

The smi21F line has a single P[lArB]-element insertion at

cytological position 21F in the isogenic Samarkand (Sam;ry506) background (Anholt et al., 1996). P-element excision

lines of smi21F were generated in the Samarkand back-

ground, as described below. Deficiency line, Df(2L)ast2/SM1, was obtained from the Bloomington Drosophila StockCenter (Bloomington, IN). All flies were reared on an agar-

yeast-molasses medium in vials maintained at 258C and

under a 12 h light/dark cycle.

Behavioral Assays

Avoidance Assay. Flies, 5–7 days posteclosion, were

removed from their food source 1.5–2 h prior to assay.

Avoidance responses to 0.3% (v/v) benzaldehyde were con-

ducted in an environmental chamber at 258C and 70%

humidity as described by Anholt et al. (1996). Briefly, one

replicate assay consisted of a single sex group of five indi-

viduals. Test vials were divided into two compartments by

placing a mark on the wall 3 cm from the bottom of the

Pinocchio Contributes to Olfaction 147

vial. The odorant was introduced on a cotton swab wedged

between the plug and the wall of the vial and the number of

flies present in the compartment remote from the odor

source was measured at 5 s intervals for 60 s. Measure-

ments began 15 s after the introduction of odorant. The

‘‘avoidance score’’ for the replicate is the average of the 10

counts, with a score of 5 indicating a maximal repellent

response. Replicate assays of each line were conducted.

Statistically significant deviations from wild-type behavior

were evaluated by ANOVA.

Attraction Assay. Olfactory trap assays were conducted

essentially as described by Woodard et al. (1989). Ten flies

of a single sex were placed in a Petri dish (100 � 15 mm)

containing 10 mL of 1% agarose and a trap constructed

from a 1.5 mL centrifuge tube and a 200 �L wide orifice

pipet tip. The tube was cut approximately 3 mm from its

end and the inverted pipette tip inserted into the tube. Three

hundred microliter of odorant in 1% agarose was applied to

the inside of the tube cap. Odorants tested were obtained

from Aldrich Chemical Co. (Milwaukee, WI) and included

acetic acid, 1-pentanol, acetone, helional, citralva, 1-buta-

nol, geraniol, cyclohexanone, amyl acetate, propanol, 3-

octanol, p-cresol, E2-hexenal, propionic acid, acetaldehyde,acetal, and ethyl propionate. After 48 h the number of flies

trapped was recorded. The ‘‘attraction score’’ is the average

number of flies trapped among replicates. Comparisons

between lines were made by three-way factorial fixed

effects ANOVA using SAS Proc GLM (SAS Institute Inc.,

Cary, NC) and posthoc Tukey tests for significant differen-

ces among means.

Electrophysiological Recordings

Electroantennogram (EAG) recordings were obtained from

Sam; ry506 (n ¼ 10) and smi21F (n ¼ 10) Drosophila 3–5

days posteclosion. Individual flies were immobilized in a

pipette tip with the anterior portion of the head protruding

from the tip. Ground and recording glass capillary electro-

des (1.5 mm outer diameter) were filled with Drosophilasaline (188 mM NaCl, 5 mM KCl, 2 mM CaCl2�2H2O). The

ground electrode was subsequently placed into the head

capsule and the recording electrode onto the anterior distal

region of the third antennal segment. A constant air stream

of �1.4 L/min flowed continuously over the antenna.

Pulses of odor consisted of 3 mL of air from a 5 cc syringe

rapidly puffed over a filter disk saturated with the diluted

odorant and then directly introduced into the air stream.

Odorants were diluted in paraffin oil (Fluka, Buchs, Swit-

zerland) and tested at three different concentrations. Each

sample was puffed two times and EAG peak amplitudes

were measured and averaged. Data were acquired using a

Syntech Intelligent Data Acquisition Controller IDAC-4-

USB (Hilversum, The Netherlands) and recording, storing,

and quantifying EAG peak amplitudes was carried out with

EAG2000 version 2.7 software (Syntech). Comparisons of

peak amplitudes between lines were evaluated by three-way

factorial ANOVA.

Quantitative Complementation Test

Deficiency line, Df(2L)ast2/SM1, which contains a deletion

over 21D1-2 to 22B2-3, was crossed to smi21F and Sam;ry506, and olfactory avoidance behavior of the F1 progeny ofeach cross assessed (Anholt et al., 1996). The data were ana-

lyzed by two-way factorial fixed effects ANOVA and quan-

titative failure to complement was inferred if the line (L) or

line by sex (S) interaction terms were significant according

to the model y¼ � þ Lþ Sþ L� Sþ E, where E indicates

error (Pasyukova et al., 2000). Significant differences

among means were determined by a posthoc Tukey test.

Phenotypic Reversion throughP-Element Excision

The P[lArB] construct was mobilized by crossing smi21Ffemales to w; Cy/Sp; SbD2-3/TM6,Tb males. Male offspring

of the genotype Sam; Cy/smiP21F; SbD2-3/ry506 were then

crossed to Sam; Cy/Sp; ry506 females and single male off-

spring in which the P-element was excised, Sam; Cy/smiP�;ry506, were again crossed to Sam; Cy/Sp; ry506 females. Male

and female Sam; Cy/smiP�; ry506 were subsequently mated

to make a homozygous P-element excision line in the iso-

genic Samarkand background. The precise excision of the

construct was verified using Southern blots of HindIII-digested genomic DNA from Sam; ry506, smi21F and P-ele-ment excision lines hybridized to a plasmid-rescued genomic

DNA fragment flanking the P-element insertion site in

smi21F. Avoidance responses to benzaldehyde of P-element

excision lines were assessed and data were analyzed using a

single classification fixed effects ANOVA according to the

model y ¼ � þ L þ E, where L designates line and E error.

mRNA Quantification

Levels of mRNA were quantified in Sam; ry506 and smi21Flines using real-time quantitative PCR. Total RNA was iso-

lated from male and female Drosophila heads using Trizol

(Gibco BRL, Gaithersburg, MD). Three replicate RNA iso-

lations were prepared for each line. The RNA was purified

by DNase treatment followed by a phenol and chloroform

extraction. cDNA was generated from 5 �g of total RNA by

reverse transcription (Stratagene, La Jolla, CA) and tran-

script-specific primers were designed by Primer Express

software (PE Applied Biosystems, Foster City, CA).

Primers and probes are as follows: transcript I, 50-CCA-CCGACAAGCTGAACTTCA-30, 50-GCAGACAGACCG-AGATTGTG-30, 6FAM-TCAGCAGCCAATATGTCG-

ATCGCCA-TAMRA; transcript II, 50-GCAGCACAG-GAATCCCAATT-30, 50-TGGCGATCGACATATTGGC-30, 6FAM-TGCTGTAAATAACCCAACAAATTGCCAT-

CAG-TAMRA. Ribosomal protein 49 (rp49) was used as

an endogenous control. Sequences for rp49 primers and

Taqman probe were: 50-CAAGAAGTTCCTGGTGCA-CAA-30, 50-AGTAAACGCGGTTCTGCATGA-30, and

6FAM-TGCGCGAGCTGGAGGTCCTG-TAMRA. The

mRNA levels were measured using the GeneAmp 5700

148 Rollmann et al.

system (PE Applied Biosystems) and the relative standard

curve method. Standard curves for rp49, CG4552, and

CG4710 transcripts I and II were constructed using dilu-

tions of cDNA from Sam; ry506. Each of the cDNA samples

was amplified in duplicate and expression levels determined

relative to the Sam; ry506 standard curve. Differences in

expression between genes in the vicinity of the P[lArB]insertion site and differences in gene expression between

smi21F and the host strain were determined by two-way

factorial ANOVA and posthoc Tukey tests, where applica-

ble. Expression of both transcripts of CG4710 was further

verified by complete sequencing of full length amplicons

corresponding to their coding regions.

In Situ Hybridization

mRNA expression patterns of the smi21F gene were deter-

mined by in situ hybridization to 10 � formalin-fixed and

paraffin-embedded sections of randomly oriented Sam;ry506 heads (Vosshall et al., 2000, with minor modification).

PCR-amplified DNA fragments corresponding to unique

portions of the coding region for each CG4710 transcript

(Fig. 3) and the neighboring gene, CG4552, were cloned

into the TA dual promotor cloning vector (Invitrogen,

Carlsbad, CA), and, following linearization of the vector,

digoxigenin-labeled antisense and sense riboprobes were

generated using SP6 and T7 RNA polymerases, respec-

tively. After deparaffinization and rehydration through

graded alcohols, slides were placed in 4% paraformalde-

hyde and subsequently washed with phosphate buffered sal-

ine (PBS). Sections were then incubated with 5 �g/mL

proteinase K for 30 min at 378C, followed by treatment

with acetic anhydride. The sections were then hybridized

for 16 h at 558C in hybridization buffer (50% formamide,

5X SSC, 5X Denhardts, 50 �g/mL yeast tRNA, 50 �g/mL

heparin, 500 �g/mL sheared salmon sperm DNA, 2.5 mMEDTA, 0.25% CHAPS, and 0.1% Tween 20) with either a

Figure 1 Behavioral responses of smi21F (white bars) and wild-type flies (Sam; ry506, blackbars) to odorants. Flies of the smi21F line show sex-specific reductions in olfactory responses to

acetic acid and propionic acid. (A) Responses of females to acetic acid, and (B) responses of males

to propionic acid. For each line, 10 (A) or 15 (B) replicates were measured for each of four odorant

concentrations.


heat denatured antisense or sense probe. After hybridiza-

tion, the slides were washed at 558C with 5X SSC followed

by 20 min sequential washes at 558C with 0.2X SSC and

PBS, 0.1% Triton X-100. Sections were then blocked with

heat inactivated normal goat serum in PBS, 0.1% Triton

X-100. Hybridization products were visualized with an

alkaline phosphatase-conjugated antidigoxigenin antibody

(Roche Molecular Biochemicals, Indianapolis, IN), using

nitro blue tetrazolium chloride and 5-bromo-4-chloro-

3-indolyl-phosphate as substrates. Images were digitally

captured and processed with Adobe Photoshop.

RESULTS

Phenotypic Characterization of thesmi21FMutant

Previously, we identified through P-element inser-

tional mutagenesis a smell impaired (smi) mutant,

smi21F, that shows odorant-specific defects in olfac-

tory avoidance behavior (Anholt et al., 1996). Aber-

rant behavioral responses were observed only in

females to benzaldehyde and 2-isobutylthiazole at

high concentrations, but not to 2-n-propylpyrazine.To further examine the specificity of the smell

impairment in this mutant, we asked whether smi21Fflies also show aberrant attractant responses to odor-

ants. Using an olfactory trap assay, we first examined

responses of the P-element free host strain, Sam;ry506, to a series of odorants, used previously by other

investigators (de Bruyne et al., 2001) and represent-

ing different structural classes, including acetic acid,

1-pentanol, acetone, helional, citralva, 1-butanol,

geraniol, cyclohexanone, amyl acetate, propanol, 3-

octanol, p-cresol, E2-hexenal, propionic acid, acetal-

dehyde, acetal, and ethyl propionate. Under our

experimental conditions [a concentration of 1% (v/v)

of each odorant] Sam; ry506 flies responded to only

five of the odorants tested, namely acetic acid, acetal-

dehyde, acetal, propionic acid, and ethyl propionate.

Although other odorants might have elicited

responses at different odorant concentrations, we

decided to focus our experiments on these five odor-

ants and generated dose-response curves for each of

them. Dose-response curves showed an increase in

attractant behavior at low odorant concentrations and

a decline at higher concentrations. We compared

attractant responses of Sam; ry506 and smi21F flies at

a range of concentrations optimal for resolving differ-

ences in attractant responses between wild-type and

mutant flies (Fig. 1). No statistically significant dif-

ferences between responses of smi21F and their wild-

type controls to ethyl propionate, acetaldehyde, and

acetal were observed (data not shown). However,

smi21F flies showed marked sex-specific reductions

in responsiveness to acetic acid and propionic acid

(Fig. 1; Table 1). The response to acetic acid was

reduced in females [Fig. 1(A); Table 1]. An apparent

shift of the dose-response toward higher stimulus

concentrations of acetic acid in the smi21F mutant is

evident from a significant difference at the intermedi-

ate concentration of 0.3% (v/v) acetic acid (p <0.0001). No statistically significant differences were

resolved at lower concentrations near threshold or at

saturating concentrations.

We also observed impaired olfactory behavior

with propionic acid [Fig. 1(B); Table 1]. In contrast

to the response to acetic acid, males, but not females,

are smell impaired at high concentrations of pro-

pionic acid, reminiscent of sexually dimorphic olfac-

tory defects observed previously for avoidance

responses to benzaldehyde and 2-isobutylthiazole

(Anholt et al., 1996). A nominally significant reduc-

tion in female response to propionic acid was also

observed at intermediate concentrations, but was not

significant after Bonferroni correction.

Table 1 Three-Way Factorial ANOVA of Behavioral Response of smi21F and Wild-Type Files to Acetic Acid and

Propionic Acidy

Acetic Acid Propionic Acid

Mean square F Value p Value Mean square F Value p Value

Line 2.3 9.11 * 5.5 17.2 ****

Sex 1.76 6.99 * 1.83 5.71 *

Treatment 9.21 36.49 *** 10.71 33.53 ****

L � S 0.001 0 ns 0.1 0.31 ns

L � T 0.79 3.12 ns 1.89 5.93 ***

S � T 0.37 1.45 ns 0.16 0.51 ns

L � S � T 0.12 0.46 ns 1.17 3.66 **

yThe model used was y ¼ � þ L þ T þ S þ L � T þ L � S þ L � T � S þ E with the three main fixed effects being line (L), treatment

(T, concentration of odorant), and sex (S), and where E indicates error. p values of <0.05, <0.01, <0.001, and <0.0001 are indicated by *, **,

***, and ****, respectively; ns, not significant.

150 Rollmann et al.

The smi21F mutant showed small, but significant

differences in its electrophysiological responses to

ethyl acetate, benzaldehyde, and ethyl propionate in

comparison to Sam; ry506, as revealed by three-way

factorial ANOVA, which showed significant variation

by line as well as line by treatment (i.e., odorant con-

centration) for all three odorants tested (Table 2).

There were also significant sex effects. Surprisingly,

for all odorants tested EAG peak amplitudes were

greater for both males and females in mutant flies

than in controls (Fig. 2; Table 2). For example, EAG

peak amplitudes for a 10�1 dilution of benzaldehyde

were 6.55 6 0.64 mV for smi21F versus 5.58 6 0.44

mV for Sam; ry506 males and 9.66 6 0.72 versus 8.01

6 0.42 mV for females (Fig. 2).

Molecular Characterization of thesmi21FMutation

To identify the candidate gene that is disrupted by the

P[lArB]-element insertion in smi21F, we analyzed

the DNA sequence flanking the P[lArB]-element

insertion site. We found that the transposon had

inserted in a predicted gene of unknown function,

designated CG4710. This gene is approximately 13.1

kb in length and generates two predicted messages of

781 and 977 bp, which we have designated as tran-

scripts I and II, respectively (Fig. 3). Both transcripts

share the last three exons, but differ in predicted tran-

scription initiation sites. The P[lArB]-element has

inserted in the second intron with respect to transcript

I, but is upstream from the transcription initiation site

in transcript II (Fig. 3). Analyses of expression of

CG4710 and another gene, CG4552, located 8.3 kb

upstream from the P[lArB] insertion site, strongly

favor CG4710 as the gene responsible for the smi21Fphenotype (see below).

To verify that the smi phenotype is indeed due to

the region of the P[lArB] insertion, we mapped the

smi21F phenotype to the P[lArB] insertion site by

quantitative complementation testing against a defi-

ciency line that contains a deletion over the 21D1-2

to 22B2-3 region, which includes CG4710. Signifi-cant variation was observed between lines [F (3, 103)

¼ 5.69; p ¼ 0.0012] and in the sex � line interaction

[F (3, 103) ¼ 3.86; p ¼ 0.012]. The deficiency, when

crossed against Sam; ry506, produced hybrid offspring

with intact olfactory avoidance behavior. However,

F1 flies derived from a cross of the deficiency with

smi21F showed smell impairment similar to the

smi21F homozygous parent. Average (6SE) avoid-

ance scores were as follows: smi21F ¼ 2.7 6 0.15

(n ¼ 28); smi21F/Df ¼ 2.7 6 0.16 (n ¼ 24); Sam;ry506 ¼ 3.3 6 0.13 (n ¼ 40); Sam; ry 506/Df ¼ 3.6 60.19 (n ¼ 19). Thus, quantitative failure to comple-

ment was observed among progeny of crosses of

smi21F and Sam; ry506 to the deficiency stock. Fur-

thermore, to verify that the smi phenotype arises fromthe P[lArB] insertion rather than as a consequence of

an unrelated mutation in the genome, we mobilized

the P[lArB]-element to demonstrate that P-element

excision can restore the wild-type phenotype. We

obtained two revertant lines from which the P[lArB]-element had excised precisely, as apparent from

Southern blot analysis. Behavioral assays

demonstrated that precise excision of the P[lArB]-construct results in restoration of wild-type avoidance

responses to 0.3% (v/v) benzaldehyde [F (2, 81) ¼0.07; p ¼ 0.93, data not shown].

The small phenotypic effect of the hypomorphic

P[lArB] insertion in smi21F, which is highly depend-

ent on genetic background as evident from extensive

epistasis (Fedorowicz et al., 1998; Anholt et al.,

2003), together with the technical limitation inherent

in the highly inbred Sam;ry506 host strain, which is

Table 2 Three-Way Factorial ANOVA of Electroantennogram Responses of smi21F and Wild-Type Files

to Ethyl Acetate, Benzaldehyde, and Ethyl Propionatey

Ethyl Acetate Benzaldehyde Ethyl Propionate

MS F Value p Value MS F Value p Value MS F Value p Value

Line 15.11 14.14 *** 4.6 6.9 * 1.09 9.07 **

Sex 97.81 91.57 **** 39.87 60.45 **** 89.54 74.34 ****

Treatment 167.7 157 **** 198.1 300.4 **** 292.7 243 ****

L � S 3.49 3.27 ns 0.25 0.37 ns 2.78 2.31 ns

L � T 5.06 4.74 ** 2.59 3.92 * 4.34 3.6 *

S � T 12.48 11.69 **** 6.75 10.24 **** 19.83 16.47 ****

L � S � T 1.22 1.14 ns 0.28 0.42 ns 0.85 0.71 ns

yThe model used was y ¼ � þ L þ T þ S þ L � T þ L � S þ T � S þ L � T � S þ E with the three main fixed effects being line (L),

treatment (T, concentration of odorant), and sex (S), and where E indicates error. p values of <0.05, <0.01, <0.001, and <0.0001 are indicated

by *, **, ***, and ****, respectively; ns, not significant.


not robust enough for transformation, precluded phe-

notypic rescue of the smi21F mutant phenotype by

introduction of a wild-type copy of the CG4710 gene.

Accumulation of deleterious mutations during more

than a decade of inbreeding has rendered the isogenic

Sam;ry506 host strain unsuitable for introduction of

transgenic constructs, because embryos of this strain

do not survive the microinjection procedure. Further-

more, the phenotypic effect of the smi21F mutation is

within the standard error of natural phenotypic varia-

Figure 2 EAG responses in the smi21F mutant and the Sam; ry506 control. (A) Typical EAG

responses of smi21F and Sam; ry506 flies illustrate the increased peak amplitudes in response to the

delivery of a 10�1 dilution of benzaldehyde to the antenna. The scale bars designate 5 mV (ordi-

nate) and 2 s (abscissa). (B,C) EAG amplitudes of male (B) and female (C) Sam; ry506 (gray bars)

and smi21F (black bars) to ethyl acetate, benzaldehyde, and ethyl propionate at dilutions of 10�5,

10�3, and 10�1. Data were analyzed by three-way factorial ANOVA, which revealed significant

variation by line and line � treatment (Table 2).

152 Rollmann et al.

tion (Mackay et al., 1996) and can only be resolved

under controlled genetic background conditions, pre-

cluding the introduction of a transgene by crossing it

from a different genetic background into the mutant

stock. Despite this limitation, the position of the P-element, expression analyses described below, and

reduction of the CG4710 transcript observed in

microarray analyses Anholt et al. (2003) strongly

implicate CG4710 as the candidate gene responsible

for the smi21F mutant phenotype. We have named

this gene of previously unknown function and its

product Pinocchio (Pino).The Pino gene encodes a 249 amino acid polypep-

tide and a 171 amino acid polypeptide corresponding

to transcripts I and II, respectively (Fig. 3). Both pol-

ypeptides are identical, except for the N-terminus,

which is 78 amino acids longer in transcript I (Fig.

4). The Pinocchio proteins contain 12 cysteines, five

of which can be aligned with characteristic cysteines

at conserved positions of OBPs (Robertson et al.,

1999; Galindo and Smith, 2001), although the overall

sequence is vastly divergent from the OBP family.

We identified orthologues of Pinocchio in the

recently sequenced Anopheles gambiae (Holt et al.,

2002) and Apis mellifera genomes (Fig. 4). It is of

interest to note that Anopheles gambiae also

expresses two transcripts corresponding to the Droso-phila Pino transcripts I and II with �40% sequence

identity, including conservation of the cysteines.

Expression of Pino Transcripts

To provide evidence that the P[lArB]-element inser-

tion in Pinosmi21F reduces gene expression we quanti-

fied mRNA levels in heads of wild-type and

Pinosmi21F flies using real-time quantitative PCR.

Transcript I was reduced 287 6 128 (SE)-fold in the

Pinosmi21F mutant (n ¼ 6), whereas transcript II

showed a 2.57 6 0.28 (SE)-fold reduction (n ¼ 6). In

contrast, the CG4552 transcript showed a 0.77 60.06 (SE)-fold difference compared to the wild-type

(n ¼ 6). To assess statistical significance the data

were analyzed by analysis of variance (Table 3).

There was a significant reduction in expression of

Pino transcript I in Pinosmi21F compared to the wild-

Figure 3 Structure of the CG4710 gene. The P[lArB] insertion site and orientations of genes and

the P-element are indicated by arrowheads and arrows, respectively. The horizontal line represents

genomic DNA in the CG4710 region. Regions of the CG4710 gene that give rise to alternative tran-scripts I and II are schematically represented. Black boxes in these regions represent coding

regions. The numerals 1 and 2 inside the squares indicate regions specific for transcript I and II,

respectively, amplified for in situ hybridizations (Fig. 5) and QPCR (Table 3).


type, while there was no significant difference in

expression of the endogenous control [Table 3(A)].

There were also significant differences in gene

expression levels when Pino transcript II, CG4552,and the endogenous control were analyzed [males,

Table 3(B), and females, Table 3(C), were analyzed

separately]. Posthoc Tukey tests (p < 0.05) indicate

that there were differences in expression in all three

genes among males with the greatest difference in

transcript II; in females, however, only differences in

expression of the Pino transcript II were significant.

These data support the notion that disruption of

Pinocchio accounts for the smell-impaired phenotype

of smi21F mutants.

If the proteins encoded by Pinocchio contribute to

olfaction, we expect Pinocchio to be expressed in

chemosensory tissues. Previously, we observed in the

Pinosmi21F mutant heterogeneous lacZ reporter gene

expression throughout the third antennal segment.

Reporter gene expression was widespread, but espe-

cially intense2 in the proximal region of the third

antennal segment (Anholt et al., 1996). In situ hybrid-

ization in Sam; ry506 flies with a riboprobe corre-

sponding to an exon of Pino transcript I reveals

extensive expression in the third antennal segments

[Fig. 5(A)], recapitulating the previously observed

lacZ reporter gene expression pattern (Anholt et al.,

1996). In contrast, we did not detect expression of

CG4552 in the third antennal segment, further corrob-

orating evidence that Pinocchio rather than the neigh-

boring gene is affected by the P[lArB]-insertion in the

smi21F mutant. We did not observe differences in

expression patterns between males and females. Stain-

ing is not observed with a sense riboprobe [Fig. 5(B)],

which shows that the staining observed with the anti-

sense probe is specific. Examination of sections

Figure 4 Amino acid sequence alignments of Pinocchio gene products with their orthologues in

the honey bee, Apis mellifera, and the malaria mosquito, Anopheles gambiae. Amino acid sequen-

ces of Pinocchio transcripts I and II were aligned with the partial sequence of the Apis melliferahmm485 protein and Anopheles gambiae XP_317770 and XP_317771 transcripts. Sequences were

aligned with the ClustalW program and alignments were refined by eye. Conserved amino acids

between Pinocchio and orthologues from the other two species are shown in red and conserved cys-

teines are shown in blue.

154 Rollmann et al.

through heads cut in various orientations and hybri-

dized to sense and antisense probes did not reveal

specific staining in the brain or in the proboscis.

DISCUSSION

Previous studies showed that the Pinosmi21F mutant

exhibits subtle odorant-specific defects in olfactory

avoidance behavior (Anholt et al., 1996). Subsequent

studies discovered that despite the relatively small

effect on olfactory behavior, the smi21F locus dis-

plays extensive epistasis with other co-isogenic smiloci (Fedorowicz et al., 1998), which led us to

hypothesize that the candidate smi gene at this locus

encodes a protein that may be required in early stages

of the process of odor recognition. Characterization

of flanking sequences of the P[lArB] insertion site,

complementation mapping, and phenotypic reversion

through P-element excision identified the CG4710gene, which we have named Pinocchio, as the candi-

date smi gene at the smi21F locus. Furthermore,

expression analysis using high-density oligonucleo-

tide microarrays showed reduction of CG4710expression (but not CG4552) in the Pinosmi21F geneticbackground (Anholt et al., 2003).

The smi21F flies show aberrant olfactory res-

ponses to benzaldehyde (Anholt et al., 1996), acetic

acid, and propionic acid (Fig. 1). We observed

increased EAG amplitudes in the smi21F mutant

upon application of benzaldehyde, ethyl acetate (used

as standard), and ethyl propionate (Fig. 2). We were

not able to test acids as they give rise to artifacts in

the EAG recordings. The increased EAG amplitude

to ethyl propionate was not correlated with an altera-

tion in the behavioral response to this odorant.

Although a strict correlation between the EAG and

expression of the behavior might not be a prioriexpected, our observations could reflect greater sensi-

tivity of the electrophysiological assay compared to

the olfactory trap assay in assessing the subtle pheno-

typic effects of this hypomorphic mutation.

Throughout our experiments we noticed concen-

tration-dependent sexual dimorphism in olfactory

impairments between Pinosmi21F males and females.

Previous studies demonstrated that the genetic archi-

tecture that underlies olfactory behavior is different

in males and females (Mackay et al., 1996; Anholt,

2004). Such sexual dimorphism can arise from differ-

ences in gene regulation in males and females or from

differences in epistatic interactions in each sex envi-

ronment (Anholt et al., 2003; Anholt, 2004; Anholt

and Mackay, 2004). Because we did not observe

overt differences in gene expression between males

and females and because epistatic interactions in

the olfactory subgenome are extensive and involve

Pinosmi21F, we favor the latter interpretation.Previously, we reported transcriptional profiles of

five coisogenic smi lines, including smi21F, and iden-

tified transregulated genes in each smi background

(Anholt et al., 2003). These studies showed extensive

transregulation of antibacterial peptides, associated

with immune defense, and biotransformation

enzymes uniquely in the Pinosmi21F genetic back-

ground (Anholt et al., 2003). In contrast, only a single

OBP transcript, Obp8a, was up-regulated in the

Pinosmi21F genetic background. These observations

and the ubiquitous expression of Pinocchio through-

out the third antennal segment, in contrast to most

conventional OBPs (Galindo and Smith, 2001), lead

us to speculate that Pinocchio may contribute to the

removal of xenobiotics, including odorants and bacte-

rial toxins, rather than odorant recognition. This

hypothesis would be in line with our observation that

EAG amplitudes are increased in the hypomorphic

Pinosmi21F mutant, possibly as a consequence of

slower odorant removal.

Pinocchio is widely expressed throughout the third

antennal segment. In addition, Pino mRNAs have also

been identified in embryonic cDNA libraries and in

adult testis. The functional significance of transcription

in these tissues remains to be evaluated. Transcrip-

Table 3 Two-Way Factorial ANOVA of pinosmi21F

Expression Levels

Mean square F Value p Value

A. pino transcript I expression levels between

smi21F and wild-type filesy

Line 457375 11.73 **

Sex 180415 4.63 ns

L � S 173794 4.46 ns

B. Male differences in expression among pinotranscript II, CG4552, and rp49{

Gene 2.33 50.77 ***

Isolation 0.03 0.63 ns

G � I 0.05 1.15 ns

C. Female differences in expression among pinotranscript II, CG4552, and rp49{

Gene 1.3 16.08 ***

Isolation 0.22 2.72 ns

G � I 0.02 0.2 ns

yNo significant difference between lines was observed for rp49.

The model used was y ¼ � þ L þ S þ L � S þ E with the two

main fixed effects being line (L) and sex (S), and{y ¼ � þ G þ I þ G � I þ E with two main fixed effects being

gene (G) and RNA isolation (I), where E indicates error. p values of<0.01 and <0.001 are indicated by ** and ***, respectively; ns,

not significant.


tional profiling studies suggest that Pinocchio may

contribute both to odorant binding and immune

defense (Anholt et al., 2003). Considering the ubiqui-

tous expression of Pino in the antenna, the odorant-

specific effects in the Pinosmi21F mutant are intriguing.

It is tempting to speculate that additional proteins in

the perilymph, possibly OBPs, may functionally inter-

act with Pinocchio. Galindo and Smith (2001) showed

differential expression of GFP-reporter genes driven

by different OBP promoters in both olfactory and

gustatory organs of Drosophila with some OBPs

showing widespread expression and others confined

to only a few cells. It is of interest to note that expres-

sion of Obp8a is up-regulated in the Pinosmi21F

genetic background (Anholt et al., 2003). A detailed

characterization of putative interactions of Pinocchio

with odorants and other components of the chemo-

sensory system in Drosophila awaits further studies.

We thank Paul Gilligan III, Nalini Kulkarni, and Monica

Mattmuller for excellent technical assistance and Yehuda

Ben-Shahar for providing sequence information for design-

ing the rp49 probes. We thank Dr. John Carlson and Rick-

ard Ignell for introducing us to EAG recording procedures

and Dr. Coby Schal for making his electrophysiological

equipment available. S. M. Rollmann is the recipient of a

Keck postdoctoral fellowship and a postdoctoral National

Research Service Award (GM20897).

REFERENCES

Anholt RRH. 2004. Genetic modules and networks for

behavior: Lessons from Drosophila. BioEssays 26:

1299–1306.

Anholt RRH, Dilda CL, Chang S, Fanara JJ, Kulkarni NH,

Ganguly I, Rollmann SM, Kamdar KP, Mackay TFC.

2003. The genetic architecture of odor-guided behavior

in Drosophila: Epistasis and the transcriptome. Nat Genet

35:180–184.

Anholt RRH, Lyman RF, Mackay TFC. 1996. Effects of

single P-element insertions on olfactory behavior in

Drosophila melanogaster. Genetics 143:293–301.

Anholt RRH, Mackay TFC. 2004. The design of genetic

analyses of complex behaviors in Drosophila. Nat Rev

Genet 5:838–849.

Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim JH,

Carlson JR. 1999. A novel family of divergent seven-

transmembrane proteins: Candidate odorant receptors in

Drosophila. Neuron 22:327–338.

Damberger F, Nikonova L, Horst R, Peng G, Leal WS,

Wuthrich K. 2000. NMR characterization of a pH-

dependent equilibrium between two folded solution con-

formations of the pheromone-binding protein from

Bombyx mori. Protein Sci 9:1038–1041.

de Bruyne M, Clyne PJ, Carlson JR. 1999. Odor coding in a

model olfactory organ: The Drosophila maxillary palp.

J Neurosci 19:4520–4532.

de Bruyne M, Foster K, Carlson JR. 2001. Odor coding in

the Drosophila antenna. Neuron 30:537–552.

Figure 5 Expression patterns of Pinocchio. Panel (A) shows expression of Pino transcript I in

cells in the third antennal segment of adult flies hybridized with an antisense riboprobe specific to

transcript I. Panel (B) shows lack of hybridization with a sense riboprobe to transcript I, demon-

strating specificity of the staining in panel (A). The second and third antennal segments are desig-

nated II and III, respectively; ‘‘s’’ designates the sacculus.

156 Rollmann et al.

Du G, Prestwich GD. 1995. Protein structure encodes the

ligand binding specificity in pheromone binding proteins.

Biochemistry 34:8726–8732.

Dunipace L, Meister S, McNealy C, Amrein H. 2001.

Spatially restricted expression of candidate taste recep-

tors in the Drosophila gustatory system. Curr Biol

11:822–835.

Fedorowicz GM, Fry JD, Anholt RRH, Mackay TFC. 1998.

Epistatic interactions between smell impaired loci in


Galindo K, Smith DP. 2001. A large family of divergent

Drosophila odorant-binding proteins expressed in gusta-

tory and olfactory sensilla. Genetics 159:1059–1072.

Gao Q, Chess A. 1999. Identification of candidate Droso-

phila olfactory receptors from genomic DNA sequence.

Genomics 60:31–39.

Gao Q, Yuan B, Chess A. 2000. Convergent projections of

Drosophila olfactory neurons to specific glomeruli in the

antennal lobe. Nat Neurosci 3:780–785.

Hallem EA, Ho MG, Carlson JR. 2004. The molecular basis of

odor coding in the Drosophila antenna. Cell 117:965–979.

Hekmat-Scafe DS, Scafe CR, McKinney AJ, Tanouye MA.

2002. Genome-wide analysis of the odorant-binding pro-

tein gene family in Drosophila melanogaster. Genome

Res 12:1357–1369.

Hekmat-Scafe DS, Steinbrecht RA, Carlson JR. 1997.

Coexpression of two odorant-binding protein homologs

in Drosophila: Implications for olfactory coding. J Neuro-

science 17:1616–1624.

Hildebrand JG, Shepherd GM. 1997. Mechanisms of olfac-

tory discrimination: Converging evidence for common

principles across phyla. Annu Rev Neurosci 20:595–631.

Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab

R, Nusskern DR, Wincker P, Clark AG, Ribeiro JM,

Wides R, et al. 2002. The genome sequence of the malaria

mosquito Anopheles gambiae. Science 298:129–149.

Horst R, Damberger F, Luginbuhl P, Guntert P, Peng G,

Nikonova L, Leal WS, Wuthrich K. 2001. NMR structure

reveals intramolecular regulation mechanism for phero-

mone binding and release. Proc Natl Acad Sci USA

98:14374–14379.

Kaissling KE. 1996. Peripheral mechanisms of pheromone

reception in moths. Chem Senses 21:257–268.

Kim MS, Repp A, Smith DP. 1998. LUSH odorant-binding

protein mediates chemosensory responses to alcohols in


Kim MS, Smith DP. 2001. The invertebrate odorant-bind-

ing protein LUSH is required for normal olfactory behav-

ior in Drosophila. Chem Senses 26:195–199.

Kruse SW, Zhao R, Smith DP, Jones DN. 2003. Structure

of a specific alcohol-binding site defined by the odorant

binding protein LUSH from Drosophila melanogaster.

Nat Struct Biol 10:694–700.

Laissue PP, Reiter C, Hiesinger PR, Halter S, Fischbach

KF, Stocker RF. 1999. Three-dimensional reconstruction

of the antennal lobe in Drosophila melanogaster. J Comp

Neurol 405:543–552.

Larsson MC, Domingos AI, Jones WD, Chiappe ME,

Amrein H, Vosshall LB. 2004. Or83b encodes a broadly

expressed odorant receptor essential for Drosophila

olfaction. Neuron 43:703–714.

Leal WS. 2000. Duality monomer-dimer of the pheromone-

binding protein from Bombyx mori. Biochem Biophys

Res Commun 268:521–529.

Leal WS, Nikonova L, Peng G. 1999. Disulfide structure of

the pheromone binding protein from the silkworm moth,

Bombyx mori. FEBS Lett 464:85–90.

Mackay TFC, Hackett JB, Lyman RF, Wayne ML, Anholt

RRH. 1996. Quantitative genetic variation of odor-

guided behavior in a natural population of Drosophila

melanogaster. Genetics 144:727–735.

Marin EC, Jefferis GS, Komiyama T, Zhu H, Luo L. 2002.

Representation of the glomerular olfactory map in the

Drosophila brain. Cell 109:243–255.

Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Men-

delsohn M, Edmondson J, Axel R. 1996. Visualizing an

olfactory sensory map. Cell 87:675–686.

Park SK, Shanbhag SR, Wang Q, Hasan G, Steinbrecht RA,

Pikielny CW. 2000. Expression patterns of two putative

odorant-binding proteins in the olfactory organs of Dro-

sophila melanogaster have different implications for their

functions. Cell Tissue Res 300:181–192.

Pasyukova EG, Vieira C, Mackay TFC. 2000. Deficiency

mapping of quantitative trait loci affecting longevity in


Pomeroy SL, LaMantia AS, Purves D. 1990. Postnatal con-

struction of neural circuitry in the mouse olfactory bulb.

J Neurosci 10:1952–1966.

Ressler KJ, Sullivan SL, Buck LB. 1994. Information cod-

ing in the olfactory system: evidence for a stereotyped

and highly organized epitope map in the olfactory bulb.

Cell 79:1245–1255.

Robertson HM, Martos R, Sears CR, Todres EZ, Walden KK,

Nardi JB. 1999. Diversity of odourant binding proteins

revealed by an expressed sequence tag project on male

Manduca sexta moth antennae. Insect Mol Biol 8:501–518.

Rubin GM. 2000. Comparative genomics of the eukaryotes.

Science 287:2204–2215.

Sandler BH, Nikonova L, Leal WS, Clardy J. 2000. Sexual

attraction in the silkworm moth: structure of the phero-

mone-binding-protein-bombykol complex. Chem Biol

7:143–151.

Scott K, Brady R Jr, Cravchik A, Morozov P, Rzhetsky A,

Zuker C, Axel RA. 2001. Chemosensory gene family

encoding candidate gustatory and olfactory receptors in

Drosophila. Cell 104:661–673.

Shanbhag SR, Hekmat-Scafe D, Kim MS, Park SK, Carlson

JR, Pikielny C, Smith DP, Steinbrecht RA. 2001. Expres-

sion mosaic of odorant-binding proteins in Drosophila

olfactory organs. Microscopy Res Technique 55:297–306.

Stocker RF. 1994. The organization of the chemosensory

system in Drosophila melanogaster: a review. Cell Tissue

Res 275:3–26.

Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB,

Axel R. 1994. Topographic organization of sensory pro-

jections to the olfactory bulb. Cell 79:981–991.

Vogt RG, Callahan FE, Rogers ME, Dickens JC. 1999.

Odorant binding protein diversity and distribution among


the insect orders, as indicated by LAP, an OBP-related

protein of the true bug Lygus lineolaris (Hemiptera,

Heteroptera). Chem Senses 24:481–495.

Vogt RG, Rogers ME, Franco MD, Sun M. 2002. A compa-

rative study of odorant binding protein genes: differential

expression of the PBP1-GOBP2 gene cluster in Manduca

sexta (Lepidoptera) and the organization of OBP genes

in Drosophila melanogaster (Diptera). J Exp Biol 205:

719–744.

Vogt RG, Rybczynski R, Lerner MR. 1991. Molecular

cloning and sequencing of general odorant-binding

proteins GOBP1 and GOBP2 from the tobacco hawk

moth Manduca sexta: comparisons with other insect

OBPs and their signal peptides. J Neurosci 11:2972–

2984.

Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R.

1999. A spatial map of olfactory receptor expression in

the Drosophila antenna. Cell 96:725–736.

Vosshall LB, Wong AM, Axel R. 2000. An olfactory sen-

sory map in the fly brain. Cell 102:147–159.

Wojtasek H, Leal WS. 1999. Conformational change in the

pheromone-binding protein from Bombyx mori induced

by pH and by interaction with membranes. J Biol Chem

274:30950–30956.

Wong AM, Wang JW, Axel R. 2002. Spatial representation

of the glomerular map in the Drosophila protocerebrum.

Cell 109:229–241.

Woodard C, Huang Sun H, Helfand SL, Carlson J. 1989.

Genetic analysis of olfactory behavior in Drosophila: a

new screen yields the ota mutants. Genetics 123:315–326.

158 Rollmann et al.

Documents

Pinocchio, a novel protein expressed in the antenna, contributes to olfactory behavior inDrosophila melanogaster