ecdysone receptor acts in fruitless- expressing neurons to mediate drosophila courtship behaviors

Ecdysone receptor acts in fruitless-expressing neurons tomediate Drosophila courtship behaviors

Justin E Dalton1,*, Matthew S Lebo1,*, Laura E Sanders1,*, Fengzhu Sun1, and Michelle NArbeitman1,2,§1Section of Molecular and Computational Biology, Department of Biological Sciences, University ofSouthern California Los Angeles, Los Angeles, California, United States of America2Section of Neurobiology, Department of Biological Sciences, University of Southern California LosAngeles, Los Angeles, California, United States of America

SummaryIn Drosophila melanogaster, fruitless (fru) encodes male-specific transcription factors (FRUM;encoded by fru P1) required for courtship behaviors [reviewed in 1]. However, downstream effectorsof FRUM throughout development are largely unknown [2-5]. During metamorphosis the nervoussystem is remodeled for adult function, the timing of which is coordinated by the steroid hormone20-hydroxy ecdysone (ecdysone) through the ecdysone receptor, a heterodimer of the nuclearreceptors EcR (isoforms are EcR-A, EcR-B1, or EcR-B2) and Ultraspiracle (USP) [reviewed in 6].Here, we show that genes identified as regulated downstream of FRUM during metamorphosis aresignificantly overrepresented with genes known to be regulated in response to ecdysone or EcR.FRUM and EcR isoforms are co-expressed in neurons in the CNS during metamorphosis in anisoform-specific manner. Reduction of EcR-A levels in fru P1-expressing neurons of males causeda significant increase in male-male courtship activity and significant reduction in size of two antennallobe glomeruli. Additional genes were identified that are regulated downstream of EcR-A in fruP1-expressing neurons. Thus, EcR-A is required in fru P1-expressing neurons for wild type malecourtship behaviors and the establishment of male-specific neuronal architecture.

Keywordssex hierarchy; fruitless; Ecdysone; courtship; behavior; glomeruli morphology

ResultsIdentification of genes regulated downstream of fru P1

Differences in transcript abundance between fru P1 mutant and wild type Drosophila maleswere examined using microarrays [7,8] in whole pupae and CNS tissues at the 48-hour afterpuparium formation (APF) stage (pupal FRUM- and CNS FRUM-regulated sets, Table S1),when FRUM is at highest abundance [9]. Transcripts from two fru P1 genotypes (fru440/P14

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NIH Public AccessAuthor ManuscriptCurr Biol. Author manuscript; available in PMC 2010 September 15.

Published in final edited form as:Curr Biol. 2009 September 15; 19(17): 1447–1452. doi:10.1016/j.cub.2009.06.063.

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and w; fruw12/ChaM5) were compared to two wild-type strains (Canton S and w Berlin,respectively). Each experiment included at least four replicates, and all showed highcorrelations and similar numbers of expressed genes (Table S2). Transcripts from 236 and 94genes show significant expression differences in the pupae or CNS, respectively (q < 0.15,moderated t-test, Table S3).

Genes in the ecdysone hierarchy are overrepresented among genes downstream of fru P1Genes functioning in the ecdysone pathway were significantly overrepresented in the gene setsidentified as regulated downstream of fru P1, based on comparisons to previously identifiedecdysone-regulated gene sets (Table 1) [10-12]. This includes genes regulated downstream ofEcR in pupae (129 genes), genes regulated downstream of ecdysone in larval organ cultures(30 genes), and genes known to function in the ecdysone pathway (8 genes) (1.4, 1.8 and 5.4fold-enrichment over what is expected at random, respectively, p < 0.0002, Tables 1,S4 andS5). Additional microarray experiments examining expression when a FRUM transgene witheither the A, B, or C DNA binding domain was over-expressed identified 156, 116, and 109genes, respectively (q < 0.05, fold-difference > 2, Table S6); each of these sets was significantlyoverrepresented with genes regulated downstream of EcR or ecdysone (Table S7) [10].

Given this observation, the presence of ecdysone receptor binding sites (EcREs) in regulatorysequence was determined for genes regulated downstream of fru P1. A significantoverrepresentation is observed, with 92/317 genes containing an EcRE (1.2 fold-enrichmentover what is expected at random, p = 0.014, hypergeometric test). The regulatory region of thefru locus contains three EcREs, which together with the observation that fru was found to beexpressed downstream of EcR in pupae [10] suggests that ecdysone receptor may regulate fruP1, ultimately regulating expression of some downstream target genes.

EcR-A and EcR-B1 are in fru P1-expressing neuronsEcR isoforms have distinct temporal and spatial expression patterns in the CNS (Figure S1)[13]. Examination of co-expression patterns of EcR-A and EcR-B1 with FRUM demonstratedthat EcR isoforms are in few FRUM-expressing cells in white pre-pupal (wpp) brains (FigureS2). By 48 hour APF and through the adult stage, no co-localization is observed between EcR-B1 and FRUM in the CNS (Figures 1 and S2). All of the examined FRUM-expressing brainneurons, and most ventral nerve cord (VNC) neurons, co-express EcR-A at 48 hour APF and0-24 hour adult stages in regions previously described (Figure 1 and S2) [9, 14]. Several cellsin the abdominal ganglion express FRUM, but not EcR-A.

Reduction of EcR function in fru P1-expressing neurons affects male courtship behaviorsEcR function was reduced in fru P1-expressing neurons and male behaviors were assayed bythe courtship index (CI) and wing extension index (WEI, Figure 2). EcR function was reducedby expressing EcR RNA interfering (UAS-IR[EcR]) or EcR dominant negative transgenes(UAS-DN[EcR]) via activation by fru P1-GAL4 (Figure 1A) [15]. Males of one experimentalgenotype showed a small, but significant reduction in male-female WEI (p < 0.05, ANOVAand post-hoc u-tests), but no flies of experimental genotypes had significant differences in CI.Males of several experimental genotypes showed significant and substantial increases in male-male WEI and CI (p < 0.05, ANOVA and post-hoc u-tests). This effect was due to reductionof EcR-A, but not EcR-B1, function. Similar results were obtained using a different fru P1-GAL4 strain (Tables S8-S9) [16]. Given that UAS-IR[EcR-A] and UAS-IR[EcR-B1] transgeneswere reported to be similarly efficacious at reducing protein levels, this suggests that EcR-Ais the predominant isoform functioning in fru P1-expressing neurons to establish wild typecourtship behaviors. Although potential functions of EcR-B isoforms cannot be ruled out, wedemonstrate EcR-A is necessary within fru P1-expressing neurons for wild type courtshipbehavior.

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Curr Biol. Author manuscript; available in PMC 2010 September 15.

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When the UAS-DN[EcR]-3 transgene was expressed to reduce EcR function within fru P1-expressing neurons of males during development, adulthood, or all stages, a significant increasein male-male courtship was observed with transgene expression during development or allstages (p < 0.05, u-tests, Figure 2G, Tables S8-S9). Reduction of EcR function throughout allstages caused higher levels of male-male courtship than reduction only during development.Male-male courtship did not significantly increase when UAS-DN[EcR]-3 expression wasrestricted to the adult stage. This indicates that the male-male courtship phenotype is primarilydue to reduction of EcR function in fru P1-expressing cells during development, and isenhanced by combined reduction during adulthood.

A study that examined males with a temperature-sensitive EcR allele demonstrated that maleflies shifted to the non-permissive temperature at adult stages displayed increased male-malecourtship [17]. The apparent difference between the results might be explained by an additionaladult requirement for EcR outside of fru P1-expressing cells. Also, the temperature-sensitiveallele of EcR might have caused developmental phenotypes in fru P1-expressing cells at thepermissive temperature, which were enhanced by shifting to the non-permissive temperatureat the adult stage.

Reducing EcR function in fru P1-expressing neurons decreases glomeruli sizefru P1-expressing olfactory receptor neurons (fru P1-ORNs) are required for the sexuallydimorphic size of antennal lobe glomeruli (DA1, VA1lm, and VL2a) [16]. Because matediscrimination has an olfactory component, EcR isoform co-expression within fru P1-ORNswas examined at 48-hour APF. All fru P1-ORNs co-express EcR-A and EcR-B1, each havingsimilar levels of expression of the respective EcR-isoform, though expression of EcR-Aappears higher than EcR-B1 throughout the antennal segment (Figures 3 and S2). All glomeruliinnervated by fru P1-ORNs in the adult are innervated by 48 hour APF (Figure 3), suggestingthat all third antennal segment fru P1-ORNs innervating antennal lobe glomeruli also expressEcR.

Antennal lobe morphology was examined in flies where fru P1-GAL4 drove expression ofUAS-EcR transgenes that reduce EcR function, and volumetric analyses were performed(Figure 3, Tables S10-S11). The volumes of two fru P1-ORN innervated glomeruli, DA1 andVA1lm, were significantly smaller when EcR-A, but not EcR-B1, function was reduced (p <0.05, ANOVA and post-hoc t-tests, Figure 3). Flies expressing the transgene causing thehighest levels of male-male courtship (UAS-DN[EcR]-3) had the smallest DA1 and VA1lmvolume, which are the two glomeruli with the largest sexual dimorphism [16]. No significantchanges in size were seen for the VL2a and VA6 glomeruli, which are also innervated by fruP1-ORNs. This is perhaps because these glomeruli do not show substantial differences involume between wild type males and females and the largest reduction in volume observedabove never results in glomeruli smaller than what is observed in females [16,18].

To determine if these decreases in glomeruli volume are due to a general requirement of EcR,and not a sex-specific effect, the volume of female glomeruli innervated by fru P1-ORNs wasanalyzed. Expression of the transgene with the largest effect in males (UAS-DN[EcR]-3)resulted in no reduction in volume (Figure 3), suggesting that in male fru P1-ORNs, EcR-A,which is not sex-specific, may act with FRUM, or some other male-specific factor, to affectglomeruli volume. The reduction of glomeruli volumes may also be due to effects from thefru P1-expressing projection neurons (PNs) innervating these glomeruli; all fru P1-expressingPNs examined express EcR-A (data not shown).

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Reduction of EcR-A in fru P1-expressing neurons affects gene expressionEcR-A levels were reduced in fru P1-expressing neurons in CNS tissues from wpp, 48-hourAPF, and 0-24 hour stages, and gene expression was examined using microarrays (EcR-A/fruP1 gene sets, Tables S1-2). Previous studies have shown that two distinct high-titer ecdysonepulses coincide with wpp and 48-hour APF stages, but not the adult stage [reviewed in 6, 19].Here, the numbers of differentially expressed genes correlate with levels of ecdysone hormone,with a higher number of genes identified during wpp and 48-hour APF stages (176 and 166genes, respectively), as compared to adults (36 genes, Figure S4, Table S12).

Significant overlap between genes expressed downstream of fru P1 and the wpp and 48-hourAPF EcR-A/fru P1 gene sets was observed (p = 0.01 and 0.019, respectively, hypergeometrictest, Figure S4). Additionally, there was significant overlap among the wpp and 48-hour APFEcR-A/fru P1 sets and previously identified sets of genes regulated downstream of EcR (Table1). The observation that more genes are regulated downstream of EcR-A during development,as compared to adult stages, is consistent with the behavioral experiments above thatdemonstrated male-male courtship phenotypes require a reduction of EcR-A function duringdevelopment.

broad (br) is directly regulated by the ecdysone receptor [reviewed in 20] and is necessary forCNS development [21-23]. Here, br expression is significantly increased in wpp CNS in maleswith reduced EcR-A function in fru P1-expressing neurons (Table S12). Co-localization of BRwith FRUM is observed in the CNS at three time points (wpp, 48-hour APF, and adult, FigureS4 and data not shown), further confirming that the ecdysone hierarchy functions in fru P1-expressing neurons during development.

DiscussionThis study demonstrates that the ecdysone hierarchy, in concert with FRUM, helps establishthe neural circuitry required to prevent male-male courtship. Genes regulated downstream ofFRUM are also regulated downstream of ecdysone or the ecdysone receptor, and contain anenrichment of EcREs in their regulatory sequence. EcR-A, but not EcR-B1, is expressed inmany FRUM-expressing neurons throughout development, and reduced EcR-A in fru P1-expressing cells results in male-male courtship and reduced glomeruli volumes.

Males with reduced EcR-A in fru P1-expressing neurons display normal courtship behaviortowards females, suggesting the neural circuitry is largely unaltered. Nevertheless, fine-scalemorphological differences may underlie the male-male courtship phenotype, consistent withthe observed reduction in volume of DA1 and VA1lm glomeruli. This difference may cause adefect in processing sensory information, such as the male-specific pheromone cis-vaccenylacetate detected by ORNs that synapse on DA1 glomeruli [24]. Here, flies of the genotype thatshowed the strongest male-male courtship phenotype showed the most substantial reductionin glomerulus volume. Identifying the causes of this male-male courtship phenotype, whichour results suggest may be due to deficits in antennal lobe glomeruli, but may also be explainedby additional sensory or higher-order processing defects, will provide insight into how neuralsubstrates that underlie complex behaviors develop.

Our data suggest that ecdysone, through EcR-A, provides temporal input to the developmentof the spatially restricted fru P1-expressing neurons, directing the precise timing of sex-specificdevelopment. EcR-A appears to primarily function in fru P1-expressing neurons during periodsof high ecdysone titers, given that the reduction of EcR-A function results in more genes withtranscriptional changes at stages of metamorphosis than the adult stage. Moreover, reductionof EcR during development, but not during adulthood, leads to increased levels of male-malecourtship.

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Three non-mutually exclusive models are proposed for how EcR and FRUM might coordinateto regulate gene expression (Figure S5). Worth noting is many of the genes identified here arelikely to be indirect targets of EcR and FRUM. Genes may be regulated downstream of the EcRand FRUM in parallel pathways (Model A). Alternatively, gene regulation may occur in linearpathways, with EcR regulating FRUM and FRUM regulating gene expression, or vice versa(Models B and C). Evidence for the model in which EcR regulates FRUM is that duringdevelopment EcR is detected in the CNS before FRUM, fru itself contains putative EcREs andfru is regulated downstream of EcR [10], whereas there is not similar data supporting FRUM

regulating EcR or usp. Further investigation of the identified genes will provide insight intohow these two independent genetic-regulatory hierarchies coordinate the large-scale changesthat remodel the nervous system during metamorphosis, setting the stage for the performanceof adult behaviors.

Experimental ProceduresSee supplemental materials.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgementsWe thank Garwin Chin, Brandon Ishaque Peter Liu, Jason Portillo and Rianna Wurzburger for their contributions tothe experiments. Maria Spletter, and Liqun Luo for assistance with glomeruli images, and Marc Green and SusanForsburg for assistance in deconvolution. We thank Eric Johnson and his lab (University of Oregon) for generouslyprinting microarrays. We are grateful to all members of the Arbeitman lab for their help. The work was funded byNIH grant 1R01GM073039 awarded to MNA.

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Figure 1. EcR isoforms have different spatial, temporal, and FRUM co-expression patterns in theCNS during development(A) EcR encodes three isoforms (EcR-A, -B1, and -B2); the isoform-specific regions (EcR-A[red], -B1 [yellow], and -B2 [green]), DNA binding domain (grey) and a ligand binding domain(blue) are indicated [adapted from 25]. UAS-IR[EcR] transgenes target the following regionsof the mRNA encoded by EcR: ligand binding domain (blue, UAS-IR[EcR]-1 and -2) [26], anEcR-A-specific portion (red, UAS-IR[EcR-A]) [27], and an EcR-B1-specific portion (yellow,UAS-IR[EcR-B1]) [27]. Transgenes that encode EcR dominant negative variants have aminoacid substitutions F645A or W650A in the ligand-binding domain (W650A: UAS-DN[EcR]-1 and -3; F645A: UAS-DN[EcR]-2 and -4]) [28,29]. The UAS-DN[EcR] transgenesreduce activity of all ecdysone receptor isoforms (UAS-DN[EcR]-1, -2, -3, and -4) [28,29]. (B-E) EcR isoform and FRUM protein localization at 48 hour APF. Immunofluorescence usingAnti-FRUM antibody (green), anti-EcR-A antibody (left panels, red) and anti-EcR-B1 antibody

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(right panels, red) in male brain (B and C) and VNC (D and E). Yellow indicates co-expressionof FRUM and EcR-A. Dashed box marks inset of P1 region of VNC. Arrow indicates abdominalganglion of VNC, where co-expression of FRUM and EcR-A is not observed in all cells. Cellswith high levels of EcR-A, presumably Type II neurons that undergo apoptosis [30], overlapwith FRUM in very few cells in prothoracic and abdominal ganglion. 20X confocal projectionsof anterior region (B and C) and 20X confocal projections of ventral region (D and E). Insetis 40X confocal projections (D).

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Figure 2. Males with reduced EcR function display high levels of male-male courtship behavior(A-G) Courtship Indices (CI, light portion of bars) and Wing Extension Indices (WEI, darkportion of bars) for male-female (A-C) and male-male courtship behavior (D-G) ofcorresponding experimental or control males (red and blue bars, respectively). (*) indicatesstatistical significance of the difference of CI or WEI of the respective genotype compared toall three controls (CS, fru P1-GAL4/+, and respective EcR transgene/+ males) at p < 0.05(Kruskal-Wallis ANOVA and post-hoc two-tailed Mann-Whitney u-tests). Numbers bracketedon right indicate the percentage of males that attempted copulation towards the male (D-F). (Aand D) Courtship of males bearing UAS-IR[EcR] transgenes designed to target commonregions of EcR mRNA. (B and E) Courtship of males containing UAS-DN[EcR] transgenes

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that affect ecdysone receptor activity in an non-isoform-specific manner. (C and F) Courtshipof males containing UAS-IR[EcR] transgenes targeting isoform-specific portions of EcRtranscripts. (G) UAS-DN[EcR]-3 restricted expression via temperature-sensitive GAL80ts

(tubP-GAL80ts) raised at restrictive (29°C, filled bars, GAL80 marked out) or permissive (19°C, non-filled bars) temperatures corresponding to pre- or post-eclosion. (†) indicates statisticalsignificance of CI or WEI of tubP-GAL80ts/UAS-DN[EcR]-3; fru P1-GAL4/+ males ascompared to CS control males p < 0.05 (two-tailed Mann-Whitney u-tests). For all genotypes,n is between 9 and 13, and data are presented as mean CI or WEI +/- SEM. See Table S8-S9for average CI and WEI and statistics.

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Figure 3. Males with reduced EcR function in fru P1-expressing neurons have reduced volumes oftwo fru P1-innervated glomeruli(A-C) Immunofluorescence of UAS-mcd8::GFP/+; fru P1-GAL4/+ males with membranebound GFP (green), anti-EcR-A (red), and anti-nc82 (purple). (A) Third antennal segment of48 hour APF pupae. Dashed box indicates region magnified in inset showing membrane boundGFP surrounding nuclear EcR-A staining. Antennal lobe of UAS-mcd8::GFP/+; fru P1-GAL4/+ male at 48 hours APF and adult (B and C, respectively). (C) Glomeruli innervated byfru P1-expressing ORNs are numbered as follows: DA1 (1), VA1lm (2), VL2a (3), and VA6(4). DA2 was used to normalize glomeruli volumes (C, arrowhead). Images are 40X confocalsections (~1 μm thick). (D) Mean of relative glomeruli volume +/- SEM of males (whitebackground) and females (grey background) is plotted on the y-axis. The additional UAStransgene that is in the genetic background, UAS-mcd8::GFP/+; fru P1-GAL4/+ (Control), is

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plotted on the x-axis. UAS-IR[lacZ] in the genetic background of UAS-mcd8::GFP/+; fru P1-GAL4/+ was used an additional control. Asterisks indicate significance (p < 0.05, one-wayANOVA and post-hoc t-tests) as compared to both Control and UAS-IR[lacZ] controlglomeruli volumes. n ≥ 10 for each genotype and glomerulus. See Table S10-S11 for averagenormalized glomeruli volumes and statistics.

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150.

2037

10.

9398

EcR

-reg

ulat

ed -1

8 to

2 h

r APF

mid

gut [

12]

170.

0608

20.

9078

190.

1747

140.

0382

20.

9972

10.

8406

61 V

erifi

ed G

enes

50.

0038

30.

0082

88.

7E-0

53

0.04

271

0.52

231

0.14

36

Num

ber o

f gen

es id

entif

ied

here

and

that

wer

e al

so id

entif

ied

in p

revi

ous e

xper

imen

ts e

xam

inin

g ec

dyso

ne a

nd/o

r EcR

-reg

ulat

ed g

ene

expr

essi

on [1

0-12

]. Si

gnifi

canc

e of

the

over

lap

was

det

erm

ined

usi

ng a

hyp

erge

omet

ric te

st. “

EcR

-reg

ulat

ed -4

to 4

hr A

PF”

refe

rs to

gene

s ide

ntifi

ed a

s dow

nstre

am o

f EcR

at -

4, 0

, and

4 h

our A

PF [1

0]. “

Ecdy

sone

-reg

ulat

ed 3

rd in

star

org

an c

ultu

re”

refe

rs to

gen

es id

entif

ied

as re

gula

ted

dow

nstre

am o

f 20-

hydr

oxye

cdys

one

in c

ultu

red

3rd

inst

ar la

rval

org

ans [

10].

“EcR

-reg

ulat

ed 0

and

5 h

r APF

mus

hroo

m b

ody”

refe

rs to

gen

es id

entif

ied

as re

gula

ted

dow

nstre

am o

f EcR

in m

ushr

oom

bod

y at

0 a

nd 5

hou

r APF

[11]

. “Ec

R-r

egul

ated

-18

to 2

hr A

PF m

idgu

t” re

fers

to g

enes

iden

tifie

d as

regu

late

d do

wns

tream

of E

cR in

mid

gut o

f -18

to 2

hou

rs A

PF [1

2]. T

he o

ther

stud

ies e

mpl

oyed

mic

roar

rays

that

incl

uded

11,

983

and

11,7

41 g

enes

that

wer

e pr

esen

t on

our a

rray

pla

tform

([10

,11]

, and

[12]

, res

pect

ivel

y). T

he “

61 v

erifi

ed g

enes

” w

ere

deem

ed e

cdys

one-

regu

late

d if

prot

ein

or m

RN

A le

vels

wer

e sh

own

in a

prim

ary

refe

renc

e to

be

ecdy

sone

-res

pons

ive

in D

roso

phila

cel

l lin

es, t

issu

es, o

r who

le a

nim

als (

Tabl

e S4

)


ecdysone receptor acts in fruitless- expressing neurons to mediate drosophila courtship behaviors

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