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Supporting InformationSun et al. 10.1073/pnas.0906377106SI
TextDrosophila Strains and Genetics. The following strains have
beendescribed previously: nan36a, iav3621, and nan-Gal4 (1);
pyx3,pyxDf4, pyxDf9, and pyxGe4; pyx3 (2); pain1, painGal4, and
UAS-pain(3, 4); GFP-nompA (5); iav-Gal4 (6), and nompC-Gal4 (6, 7).
Inparticular, the nompC-Gal4 construct contains a 2-kb
genomicfragment that is 5� to the translational start site of
nompC. FivenompC-Gal4 transgenic lines were analyzed for expression
inJohnston’s organ; line No. 25 (described as nompC-GAL4.25 inref.
7) showed the most extensive expression in Johnston’s organneurons
and was selected for this study. Another line showedoverlapping but
less extensive expression, and the rest of the linesshowed no
detectable expression in Johnston’s organ. ThenompCf00642 strain
was obtained from the Exelixis Collection atHarvard (line f00642)
and backcrossed to the w1118-WLS strain for6 generations for the
tube-climbing test. The resulting line wasfurther backcrossed to
the w2202u strain for 6 generations withreplacement of the X
chromosome of Canton-S (2202u) andtested in the vertical choice
maze (Note S1). The pain1 andpainGal4 strains congenic with
Canton-S (2202u) were kindlyprovided by T. Kitamoto (University of
Iowa). The pyx3 straincongenic with Canton-S (2202u) was obtained
by backcrossingthe original pyx3 strain to the w2202u strain for 8
generations andthen replacing the X chromosome of Canton-S (2202u).
UAS-GFP, UAS-mCD8::GFP, UAS-Nuclear DsRed (RedStinger),
andUAS-myr-mRFP were obtained from the Drosophila stock centerat
Bloomington, IN. Appl-Gal4 is a gift from R.S. Hewes(University of
Oklahoma).
A 1-kb genomic DNA sequence that was 5� to the
translationalstart site of pyx was cloned into the pPTGAL vector
(8) and usedto make the transgenic line pyx-Gal4. A cDNA clone
(AT05393)of the pyx-PA transcript was obtained from Drosophila
GenomeResource Center. The N717, L718, and M719 residues encodedby
this cDNA clone were changed to F717, A718 and P719 (NoteS3) with
the QuikChange II kit (Stratagene) and the primer
pair:5�-GGTAATTTTAACCTTTGCCCCGGTGGGATTGGCC-G-3� and
5�-CGGCCAATCCCACCGGGGCAAAGGTTA-AAATTACC-3�. The resulting pyxFAP
sequence was subclonedinto the pUAST vector and used to make the
transgenic lineUAS-pyxFAP.
Histochemistry and Microscopy. To image a whole antenna,
theprefrons cuticle with the antennae attached was dissected
fromthe head and fixed in 4% paraformaldehyde at room tempera-ture
for 30 min. The specimens were washed in PBS and mountedon slides
with Vectashield mounting medium H-1000 (VectorLaboratories). For
better resolution of cell types and finestructures in Johnston’s
organ, frontal sections of the antennawere cut at 10-�m thickness
with a cryostat equipped with theCryoJane Tape-Transfer system
(Instrumedics). Specimen prep-aration before sectioning was
performed according to Protocol13.2 of ‘‘Drosophila Protocols’’
(9). Antibody staining wasperformed with standard techniques. Actin
filaments in scolo-pale rods were stained with Alexa Fluor 633
conjugated phal-loidin (Invitrogen, Cat. No. A22284) according to
manufactur-er’s instructions. The brain and thoracic ganglia were
dissectedand immunostained according to a standard protocol
(10).
All images were taken with an Olympus Fluoview FV1000confocal
microscope equipped with differential interferencecontrast (DIC).
The entire Johnston’s organ was visualized inZ-stacks with the step
size set at 0.9 �m; laser intensity and signalgain were compensated
for deeper Z slices with the BrightZ
function of the FV10-ASW software. Z-projections and
3-Dprojections of Z-stacks were also accomplished with FV10-ASW. In
Fig. 4 B and C, we used the volume viewer function ofImageJ to
visualize the nc82 stained brain; the mCD8::GFPsignal was overlaid
on that image.
RT-PCR and Real-Time PCR Assays. Flies were separated by
genderunder CO2 and flash frozen in liquid nitrogen. Total RNA
fromwhole flies was extracted with RNA STAT-60TM
(TEL-TEST)according to manufacturer instructions. The resulting
total RNAwas further purified with an RNeasy mini kit (Qiagen)
andtreated with RNase-free DNase set (Qiagen) to remove
residualgenomic DNA. Reverse transcription (RT) was performed
byusing the TaqMan reverse transcription reagents (Part No.N8080234
Applied Biosystems) with 0.2 �g total RNA in a 20-�Lreaction. PCRs
were performed with the TaqDNA polymerase(Roche) in 50-�L reactions
and were subsequently analyzed ona 0.8% agarose gel. The primers
used to check mRNA splicingaround the f00642 piggyBac insertion
were 5�-GCAACGAAG-GACAATAAGAC-3� (forward) and
5�-CATTCGTTCCGTA-ATCAACC-3� (reverse). Real-time PCR assays were
performedon an ‘‘ABI7500 fast’’ machine with the PowerSybr
reagentaccording to manufacturer instructions. The primers used
tospecifically detect the amplicon representing correct
splicingbetween the exon 3 and exon 4 junction of nompC
were:5�-ACCGGTGGCTCGCGTT-3� (forward) and
5�-ATTAGTT-GCAGTTCCGGTTTGTC-3� (reverse). Expression levels of18s
rRNA were used as an RNA-loading control. The primers for18s rRNA
were provided in the TaqMan ribosomal RNA controlreagents (Part No.
4308329, Applied Biosystems). Data weretransformed according to the
� � Ct method and are representedas relative values.
Statistical Analysis. Quantitative results are presented as mean
�SEM. Unpaired t tests were performed for 2-group comparisons.ANOVA
followed by post hoc test of Games–Howell wasperformed with SPSS-17
for multiple comparisons among morethan 2 groups. The Games–Howell
test controls for unequalvariances and unequal sample sizes among
the groups.
Supplemental Notes. Note S1. Among the TRP mutant
lines,nompC-null f lies show very poor viability, and the few
survivingadults are severely uncoordinated (11). These phenotypes
arelikely because of an overall inactivation of
mechanosensorybristles. They precluded us from using nompC-null f
lies to assessthe specific contribution of nompC to the
gravity-sensing func-tion of Johnston’s organ. Therefore, we
characterized a nompCmutant line which has normal viability as
homozygous adults andis therefore suitable for geotaxis behavioral
assays. This mutantis line f00642 in the Exelixis collection of
piggyBac insertions. Wenamed the mutant allele nompCf00642.
nompCf00642 harbors asingle piggyBac insertion within the third
intron of the nompCgene. By RT-PCR assays (see Materials and
Methods), we foundthat the insertion disrupted splicing of nompC
premRNA; as aresult, the amount of correctly spliced mRNA is
reduced by�90% (Fig. S2). Consistent with the gene expression
changes, wefound a remarkable hearing defect in the mutant; the
amplitudeof sound-evoked antennal responses was reduced by �60%
innompCf00642 f lies compared with wild-type controls (Fig. 6B).The
severity of this hearing deficit is comparable with publishednompC
null mutants (12), suggesting that nompCf00642 is a strongmutant
allele in terms of affecting the mechanosensory function
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of Johnston’s organ. One limitation for the use of this line is
thatour RT-PCR assays were done using total RNA from whole
flies.Thus, we do not know whether the f00642 insertion
affectsnompC splicing equally in all subpopulations of Johnston’s
organneurons, i.e., those involved in geotaxis as opposed to
hearing (7,13).
Initially, we found that the isogenic w1118 control (14)
(Bloom-ington stock # 6326) and the nompCf00642 mutant were
bothdefective in the anti-gravity climbing assay (Fig. S3).
Tworandomly picked Exelixis piggyBac insertion lines (f06539
ande02329, isogenic with stock #6326) were also defective in the
test(Fig. S3). However, a different w1118 stock maintained in our
lab,which we refer to as w1118-WLS, is normal in the tube-climbing
test(Fig. 3 A and B and Fig. S3). The w1118-WLS stock was
originallyacquired from Dr. Wayne A. Johnson (University of Iowa).
Inthe name w1118-WLS, ‘‘WLS’’ stands for Welsh Lab Strain.
Ourobservations indicate that variations in genetic background
mayhave a significant impact on negative geotaxis, even if
thevariations are between 2 lab stocks designated the same.
Toresolve the genetic background issue, we backcrossed the
orig-inal nompCf00642 line to the w1118-WLS line for 6 generations.
Thechange in nompC mRNA splicing is retained in
backcrossednompCf00642 f lies (Fig. S2), but their behavior in the
tube-climbing test is normal and indistinguishable from w1118-WLS f
lies.We also examined the behavioral effect of nompCf00642 in
thevertical choice maze assay. Because the w- mutation in
thew1118-WLS background may result in aberrant behavior in
thisassay (15), we further backcrossed nompCf00642 to
Canton-S(2202u) with replacement of the w� X chromosome. In
thisgenetic background, nompCf00642 did not affect behavior in
thechoice maze (Fig. S1). These results, together with the
hearingdefects we found with the nompCf00642 mutation, suggest that
inJohnston’s organ the NompC channel mediates sound detection,but
not gravity sensing.Note S2. nompCf00642, pain1, painGal4 and pyx3
mutants had normalscores in the Light condition of the climbing
assay (Fig. 3A),indicating that their general locomotion capability
was intact. Incontrast, nan36a and iav3621 mutants had reduced
scores in the
Light condition, a sign of impaired general locomotion (Fig.
3A).nan36a and iav3621 f lies were also uncoordinated when passing
theT-shaped choice points in the vertical maze. These
observationsare consistent with previous descriptions of nan and
iav mutants(1, 16) and may be due to disrupted function of
femoralchordotonal organs thought to mediate proprioception
(17).Defective femoral chordotonal organ function may also
explainwhy nan36a and iav3621 mutants performed worse in the
behav-ioral tests than flies with the glue treatment that
selectivelyaffects Johnston’s organ.Note S3. The transgene pyxFAP
contains the full-length cDNA ofpyx-RA and carries the following
mutations introduced by site-directed mutagenesis: N717F, L718A,
and M719P. The 3 aminoacids are located in the sixth transmembrane
domain of Pyx andare conserved among all 4 Drosophila TRPA
proteins. Previouswork on mammalian TRP channels showed that
mutating con-served residues in the sixth transmembrane domain
yields dom-inant-negative channel subunits (18, 19).Note S4. To
rotate the fly body during electrophysiologicalrecordings, the
experimenter moves a handle on the edge of theapparatus by hand.
Pushing or pulling the handle results in asmooth rotation without
noise. The only audible sound occurs atthe end of each rotation
when the wires associated with the headstage contact the supporting
platform of the apparatus. This softsound is unlikely to have a
significant effect on the fly antennafor several reasons. First,
the fly antenna is only sensitive tonear-field sound, that is, bulk
movement of air particles close tothe sound source. Away from the
source, energy of the movingair particles is inversely correlated
with square of the distance(20). The sound of a wire tinkling
several inches away from thefly would have essentially no
near-field energy at the fly. Second,the sound is transient and
occurs only at the end of the rotationand not at the beginning. In
contrast, the train of spikes typicallyinitiates when the rotation
starts and lasts until the end of therotation. Third, in control
experiments we shielded the fly witha Plexiglas cage. Any
significant source of near-field soundshould have also been blocked
by the cage. Because the antennaresponded the same with or without
the cage, there is likely nosignificant auditory component involved
in the recording.
1. Kim J, et al. (2003) A TRPV family ion channel required for
hearing in Drosophila.Nature 424:81–84.
2. Lee Y, et al. (2005) Pyrexia is a new thermal transient
receptor potential channelendowing tolerance to high temperatures
in Drosophila melanogaster. Nat Genet37:305–310.
3. Al-Anzi B, Tracey WD, Jr, Benzer S (2006) Response of
Drosophila to wasabi is mediatedby painless, the fly homolog of
mammalian TRPA1/ANKTM1. Curr Biol 16:1034–1040.
4. Tracey WD, Jr, Wilson RI, Laurent G, Benzer S (2003)
painless, a Drosophila geneessential for nociception. Cell
113:261–273.
5. Chung YD, Zhu J, Han Y, Kernan MJ (2001) nompA encodes a
PNS-specific, ZP domainprotein required to connect mechanosensory
dendrites to sensory structures. Neuron29:415–428.
6. Liu L, et al. (2007) Drosophila hygrosensation requires the
TRP channels water witchand nanchung. Nature 450:294–298.
7. Kamikouchi A, et al. (2009) The neural basis of Drosophila
gravity-sensing and hearing.Nature 458:165–171.
8. Sharma Y, Cheung U, Larsen EW, Eberl DF (2002) PPTGAL, a
convenient Gal4 P-elementvector for testing expression of enhancer
fragments in Drosophila. Genesis 34:115–118.
9. Sullivan W, Ashburner M, Hawley RS (2000) in Drosophila
Protocols (Cold Spring HarborLaboratory Press, Plainview, NY), p
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10. Wu JS, Luo L (2006) A protocol for dissecting Drosophila
melanogaster brains for liveimaging or immunostaining. Nat Protoc
1:2110–2115.
11. Kernan M, Cowan D, Zuker C (1994) Genetic dissection of
mechanosensory transduc-tion: Mechanoreception-defective mutations
of Drosophila. Neuron 12:1195–1206.
12. Eberl DF, Hardy RW, Kernan MJ (2000) Genetically similar
transduction mechanisms fortouch and hearing in Drosophila. J
Neurosci 20:5981–5988.
13. Yorozu S, et al. (2009) Distinct sensory representations of
wind and near-field sound inthe Drosophila brain. Nature
458:201–205.
14. Parks AL, et al. (2004) Systematic generation of
high-resolution deletion coverage ofthe Drosophila melanogaster
genome. Nat Genet 36:288–292.
15. Armstrong JD, Texada MJ, Munjaal R, Baker DA, Beckingham KM
(2006) Gravitaxis inDrosophila melanogaster: A forward genetic
screen. Genes Brain Behav 5:222–239.
16. Gong Z, et al. (2004) Two interdependent TRPV channel
subunits, inactive and Nan-chung, mediate hearing in Drosophila. J
Neurosci 24:9059–9066.
17. Kernan MJ (2007) Mechanotransduction and auditory
transduction in Drosophila.Pflugers Arch 454:703–720.
18. Kuzhikandathil EV, et al. (2001) Functional analysis of
capsaicin receptor (vanilloidreceptor subtype 1) multimerization
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Clapham DE (2006) The TRPM7ion channel functions in cholinergic
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Fig. S1. The vertical choice maze assay. (A) The geotaxis choice
maze. The red arrow indicates direction of gravity. The white arrow
points to the maze entrance.The yellow arrows indicate direction of
light. Exit positions are numbered 1–9 with 1 at the bottom and 9
at the top. (B) Distribution of control CS flies (n � 137,n refers
to the total number of flies that finished the maze) and CS flies
(n � 155) with glued antennae at the exits of the maze. Values on y
axis indicate percentageof flies at each exit. MMEV is mean maze
exit value (15). *, P � 0.05 by unpaired t test. (C) nompCf00642
(backcrossed to CS, n � 203) and CS control (n � 217) weretested in
parallel in the vertical choice maze. P � 0.54 by unpaired t test
for MMEV. (D) pain1 (backcrossed to CS, n � 226) and CS control (n
� 235) were testedin parallel in the vertical choice maze. *,
significant difference from the control, P � 0.05 by unpaired t
test of MMEV. (E) pyx3 (in its original genetic background,n � 116)
and a control strain expressing the pyxGe transgene that rescues
pyx3 (n � 135) were tested in parallel in the choice maze. *, the
mutant was significantlydifferent from the genetic rescue group, P
� 0.05 by unpaired t test of MMEV. (F) nan36a (n � 103) and iav3621
(n � 93) were tested in parallel in the choice maze.The exit of
these mutants at the bottom of the choice maze is likely due to
their mobility defect - poor coordination when passing the T-shaped
intersections.The vertical choice maze method. Flies were raised
and collected into groups of 25 in the same conditions as described
for the tube-climbing test. The choicemaze was built with T-shaped
and Y-shaped connectors and segments of polypropylene tubing,
according to the specifics described in ref. 15 except that
theconnector arms were not shortened. Two choice mazes were set
vertically and in parallel in a box made of cardboard. The box had
2 openings. The small openingon the back side allowed access to the
maze entrance. The slit on the front side allowed all collection
tubes of both mazes to protrude out of the box. A 63.5-mm,34-W
fluorescent strip lamp was set vertically facing the collection
tubes. Flies were transferred and released into the maze 1 by 1.
The number of flies in eachcollection tube was counted 2 h later.
For most genotypes, a 2-h test period allowed �80% of flies to exit
the maze. Because nan36a and iav3621 flies had reducedmobility
(Note S2), we extended the test period to 3 h.
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Fig. S2. Abnormal splicing of nompC mRNA in nompCf00642 flies.
(A) Three transcripts annotated in FlyBase (version R5.11 for
Drosophila melanogaster). ThepiggyBac insertion f00642 is located
in the third intron which is shared by all 3 transcripts. (B)
RT-PCR around the insertion site of f00642. The location of
theprimer pair with respect to nompC transcripts is shown by red
half arrows in A. m/m refers to homozygous nompCf00642 flies, m/�
refers to heterozygousnompCf00642 flies, and �/� refers to
wild-type w1118 controls. The size of Band I is �850 bp, Band II is
�780 bp, and Band III is �480 bp, which represents the
productexpected in wild-type flies. N.S. refers to a nonspecific
PCR product. (C) Quantitative RT-PCR (qRT-PCR) experiments to
assess the amount of correctly splicednompC mRNA in nompCf00642
flies relative to that in wild-type controls. Each row in the table
represents an independent experiment. #6326 refers to
Bloomingtonstock #6326, a w1118 strain. WLS refers to Welsh Lab
Strain of w1118. iso indicates isogenic. (D) Fragments A–C are the
parts of the piggyBac transposon that areaberrantly spliced into
nompC mRNA. These fragments were identified by cloning the aberrant
PCR products shown in B and DNA sequencing. Numbers inparentheses
denote the boundaries of each fragment with respect to the full
sequence of the piggyBac construct (GenBank Accession No.
AY515148). SA indicatessplice acceptor-like sequence and SD
indicates splice donor-like sequence. Band I in B contains
fragments A and B, Band II in B contains just Fragment B, and
BandIII in B is wild-type containing none of the piggyBac
fragments. The combination of Fragments A and C also exists in
aberrantly spliced nompC RNA, but it doesnot show up as a specific
band in B probably because of low abundance. The table shows that
the 3 types of aberrant splicing events introduce a premature
stopcodon in the mRNA.
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Fig. S3. The genetic background of the Bloomington #6326 line
(w1118) causes defective negative geotaxis. The #6326 strain
(w1118) in the Bloomington StockCenter was impaired in anti-gravity
climbing in the Dark condition. The nompCf00642 line (n � 11
trials) plus 2 randomly chosen lines [f06539 (n � 16 trials)
ande02329 (n � 14 trials)] of the Exelixis collection are isogenic
to #6326 (n � 9 trials) and showed similar impairment. ANOVA showed
no significant differenceamong the 4 isogenic lines (P � 0.20). The
w1118-WLS line (n � 14 trials) showed negative geotaxis and the
nompCf00642 mutation (n � 27 trials) did not impairnegative
geotaxis when it was placed in the w1118-WLS background through 6
generations of backcrossing. Parts of the results in Fig. 3 are
displayed here againfor comparison.
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Fig. S4. The experimental setup for recording the
electrophysiological response of Johnston’s organ to body
rotations. (A) Picture of the setup. Key elementsare labeled with
numbers: 1, axis of rotation (dashed line); 2, aluminum platform;
3, micromanipulator; 4, head stage of amplifier; 5, recording
electrode; 6,mounting of fly in plastic pipette; 7, reference
electrode and wire; and 8, handle. (B) Demonstration of a 90°
pitch.
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Table S1. The TRP family genes and mutations in this study
TRP gene (abbreviation) TRP subfamily Mutation name Mutation
type
no mechanoreceptorpotential C (nompC)
TRPN nompCf00642 mRNA splicing disrupted by piggyBac
transposon(Note S1 and Fig. S2 and Fig.S3)
painless (pain) TRPA pain1 Transcription and mRNA splicing
disrupted by P-element in 5�UTR (1)painGal4 A modified P-element
replacing the P-element in pain1 (1)
pyrexia (pyx) TRPA pyx3 Loss of mRNA and protein caused by
P-element insertion in exon (2)pyxDf9 Loss of Transcript RA (long
form) caused by imprecise excision
of P-element (2)pyxDf4 Loss of Transcript RB (short form) caused
by imprecise excision
of P-element (2)nanchung (nan) TRPV nan36a Loss of mRNA and
protein caused by P-element local hopping (3)inactive (iav) TRPV
iav3621 Loss of protein caused by chemically induced genomic
deletion (4)
1. Tracey WD, Jr., Wilson RI, Laurent G, Benzer S (2003)
Painless, a Drosophila gene essential for nociception. Cell
113:261–273.2. Lee Y, et al. (2005) Pyrexia is a new thermal
transient receptor potential channel endowing tolerance to high
temperatures in Drosophila melanogaster. Nat Genet 37:305–310.3.
Kim J, et al. (2003) A TRPV family ion channel required for hearing
in Drosophila. Nature 424:81–84.4. Gong Z, et al. (2004) Two
interdependent TRPV channel subunits, inactive and Nanchung,
mediate hearing in Drosophila. J Neurosci 24:9059–9066.
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Movie S1 (MOV)
Movie S1. The 3-D animation shows that painGal4 and pyx-Gal4
drove Nuclear DsRed expression in 2 populations of nuclei in
Johnston’s organ. The spatialdistribution of these nuclei resembles
2 concentric rings: the outer ring corresponded to painGal4, and
the inner ring corresponded to pyx-Gal4. The green axispoints from
the ventral to the dorsal side of the fly body. The blue axis
points roughly from posterior to anterior. The red axis points
roughly from medial to lateral.
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