Open Access

A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis

  • Kimberly Regna1,
  • Peri T. Kurshan2, 5,
  • Benjamin N. Harwood2,
  • Adam M. Jenkins1,
  • Chao-Qiang Lai3,
  • Marc A.T. Muskavitch1, 4,
  • Alan S. Kopin2 and
  • Isabelle Draper2Email author
Contributed equally
BMC Developmental BiologyBMC series – open, inclusive and trusted201616:15

https://doi.org/10.1186/s12861-016-0115-z

Received: 11 November 2015

Accepted: 8 May 2016

Published: 16 May 2016

Abstract

Background

Insect metamorphosis relies on temporal and spatial cues that are precisely controlled. Previous studies in Drosophila have shown that untimely activation of genes that are essential to metamorphosis results in growth defects, developmental delay and death. Multiple factors exist that safeguard these genes against dysregulated expression. The list of identified negative regulators that play such a role in Drosophila development continues to expand.

Results

By using RNAi transgene-induced gene silencing coupled to spatio/temporal assessment, we have unraveled an important role for the Drosophila dopamine 1-like receptor, Dop1R2, in development. We show that Dop1R2 knockdown leads to pre-adult lethality. In adults that escape death, abnormal wing expansion and/or melanization defects occur. Furthermore we show that salivary gland expression of this GPCR during the late larval/prepupal stage is essential for the flies to survive through adulthood. In addition to RNAi-induced effects, treatment of larvae with the high affinity D1-like receptor antagonist flupenthixol, also results in developmental arrest, and in morphological defects comparable to those seen in Dop1R2 RNAi flies. To examine the basis for pupal lethality in Dop1R2 RNAi flies, we carried out transcriptome analysis. These studies revealed up-regulation of genes that respond to ecdysone, regulate morphogenesis and/or modulate defense/immunity.

Conclusion

Taken together our findings suggest a role for Dop1R2 in the repression of genes that coordinate metamorphosis. Premature release of this inhibition is not tolerated by the developing fly.

Keywords

Development Dopamine GPCR RNAi Transcriptome

Background

The naturally occurring catecholamine dopamine (DA) acts as a neurotransmitter and neurohormone in the central nervous system (CNS) of vertebrates and invertebrates. DA is a precursor in the biochemical pathway for the production of melanin, and is required for invertebrate cuticle sclerotization [14]. Increasing evidence suggests that in insects, DA and DA receptors (DARs) are involved in the regulation of larval and pupal ecdysis, as well as in metamorphosis [59].

DA metabolism has been studied extensively within many phylogenetic groups. The essential steps required for dopaminergic neurotransmission (i.e., DA synthesis, release, receptor activation, and reuptake) are conserved between flies and humans. DA synthesis is controlled by the rate-limiting enzyme tyrosine hydroxylase (TH), which is encoded in Drosophila by the pale locus [14, 10]. TH converts tyrosine to the precursor molecule L-DOPA, which is in turn converted to DA by the enzyme DOPA decarboxylase (DDC), encoded by the Ddc gene [59, 11, 12]. TH and DDC are required for normal development in Drosophila. Null mutations targeting either biosynthetic enzyme result in late embryonic lethality [13, 14]. More recently, elegant studies have shown that selective depletion of TH in the nervous system is well tolerated by the developing fly, and that corresponding adults have normal lifespan, albeit display behavioral deficits (e.g., motor activity, phototaxis, aversive learning) [15].

DA exerts its function by activating G protein-coupled receptors (GPCRs). The fruit fly expresses both D1-like and D2-like DA receptors, which are distinguished based on the ability of the receptor to couple to either stimulatory Gαs (D1-like) or inhibitory Gαi/o (D2-like) G proteins, which in turn activate downstream signaling mechanisms [16]. The fly D1-like receptors include Dop1R1 (synonyms: DopR1, dDA1, dumb, Dmdop1, DA1) [17, 18] and Dop1R2 (synonyms: DopR2, DAMB, DOPR99B) [19, 20], as well as the non-canonical DopEcR (synonym: dmDopEcR) [2123]. DopEcR has a unique in vitro pharmacological profile and can be activated either by dopamine or by the steroid hormone 20-hydroxyecdysone (20E) [22]. There is only one known Drosophila D2-like receptor, Dop2R (synonym: DD2R, D2R), which has also been cloned and characterized [24].

In addition to modulating a range of receptor-mediated physiologies in insects [2533], DA acts as a precursor of metabolites involved in cuticle melanization (pigmentation) [4], and is essential for the crosslinking of proteins and chitin during sclerotization (hardening) of the cuticle after eclosion [2, 3437]. Although the importance of DA GPCRs as modifiers of adult fly behavior (including locomotor activity, memory, arousal, temperature preference, courtship, gustation, olfaction and response to drugs of abuse) is well-documented [21, 23, 2531, 38, 39], the contribution of DA receptors in the modulation of developmental processes has remained poorly defined. DopEcR, which responds to both DA and ecdysone, has been shown to regulate sugar sensing, male courtship, and pheromone perception in adult insects [22, 23, 40]. Overexpression or a significant reduction in the expression of this receptor, however, does not compromise normal development [22]. The focus of our study is to define the role of the D1-like Drosophila DA receptor, Dop1R2, during development. This GPCR is well-conserved in arthropods, but exhibits limited homology with mammalian dopamine receptors [41, 42], suggesting a unique function for Dop1R2 that is specific to invertebrate physiology.

We have used transgenic Dop1R2 RNA interference (RNAi) Drosophila, and characterized the effects of Dop1R2 knockdown (KD) using the GAL4/UAS-mediated system. We demonstrate that Dop1R2 activity is critical during the third larval instar and pupal stages to ensure completion of development through adult emergence. Our investigations of the tissue/cell types that underlie the observed Dop1R2-mediated phenotypes suggest the involvement of Dop1R2 receptors expressed in the salivary glands. The Dop1R2 RNAi-induced phenotypes observed in escaper adults are recapitulated in progeny exposed to a Dop1R2 small molecule antagonist. We have identified a subset of genes that respond to Dop1R2 KD, and are essential in development. Our data provide the first indications that a peripheral dopamine receptor controls key developmental processes in Drosophila.

Results

Dop1R2 RNAi flies exhibit decreased Dop1R2 transcript levels

Crossing UAS-dsDop1R2 RNAi transgenic flies with the Act5C-GAL4 driver strain (Fig. 1a and b) results in progeny that ubiquitously express Dop1R2 double-strand (ds) RNA (Act5C is the cytoskeletal actin 5C). This leads to targeted degradation of the endogenous Dop1R2 mRNA (i.e., Dop1R2 “knockdown”, or KD) in all tissues in which the receptor is normally expressed (Fig. 1c). When primers were designed to amplify the endogenous Dop1R2 message, without amplifying the RNAi sequence, a significant and reproducible decrease in Dop1R2 expression was observed, in Dop1R2 RNAi vs. control flies (Fig. 2a and b). When PCR primers were designed to amplify the Dop1R2 RNAi construct, a marked increase in transcript level was observed, confirming the expression of the RNAi transgene (Fig. 2b). To assess whether expression of the Dop1R2 RNAi construct could trigger off-target effects, expression of a series of other biogenic amine receptors with closest homology (36–43 % identity as assessed at the nucleotide level via ClustalW alignment [43] with Dop1R2 were also assayed. These included the second fly dopamine D1-like receptor Dop1R1, the dopamine D2-like receptor Dop2R, the octopamine receptor Oamb, the tyramine receptor Oct-TyrR and the serotonin receptor 5-HT1A. There was no significant change in the expression level of each GPCR gene under study in Dop1R2 RNAi vs. control flies, except for that of Dop2R (the D2-like dopamine receptor), for which a slight increase was observed (Fig. 2b).
Fig. 1

Dop1R2 cDNA and a corresponding interference construct. a Dop1R2 alternative transcripts Dop1R2-RA, Dop1R2-RB and Dop1R2-RC are shown. Top: coding sequences encompassing transmembrane domains (TMDs) 1-7 are shaded (dark gray boxes) and UTR regions (light gray boxes). Bottom: knockdown region expanded, with TMDs 5-7 indicated (white boxes). b pUAS-dsDop1R2 interference construct, including the yeast Upstream Activator Sequence (UAS; binding site for the yeast transcription factor, GAL4), the Dop1R2 inverted repeats and an SV40 polyadenylation site. c Crosses and knockdown schematic, including Dop1R2 inverted repeat (black)

Fig. 2

Dop1R2 RNAi flies show decreased Dop1R2 transcript levels. a Transcript levels assessed by RT-PCR. RNA from Dop1R2 RNAi and control flies was reverse transcribed, and PCR was performed in triplicate using primer sets corresponding to endogenous Dop1R2 as well as other biogenic amine receptors (such as Dop1R1, shown as a reference). b Dop1R2 transcript levels are significantly decreased in Dop1R2 RNAi flies (genotype: w1118;UAS-dsDop1R2/+;Act5C-GAL4/+), compared to controls (genotype: w1118;UAS-dsDop1R2/+;TM6B/+). The average band intensity of the Dop1R2 RNAi PCR product was compared to that of control PCR product for the same gene. Primers corresponding to Dop1R2 as well as other biogenic amine receptors (Oamb, octopamine receptor; Oct-Tyr, tyramine receptor; 5-HT1A, serotonin receptor 1A; Dop1R1, other D1-like Dopamine receptor; Dop2R, D2-like dopamine receptor) and an Actin5C control were used (Additional file 14: Table S1). The difference in PCR band intensity for the assessed genes (in Dop1R2 RNAi vs. control flies) are as follows: Dop1R2 (mRNA, in/out primers): -8.6; Dop1R2 (RNAi construct, in/in primers): 20.9; Oamb: -1.2; Oct-TyrR: 0.5; 5-HT1A: -1.1; Dop1R1: -0.1; Dop2R: 3.0; Act5C: -1.3. Symbols: Dop1R2 (Dopamine 1-like receptor 2, CG18741), Oamb (Octopamine receptor in mushroom bodies, CG3856), Oct-TyrR (Octopamine-Tyramine receptor, CG7485), 5-HT1A (5-hydroxytryptamine (serotonin) receptor 1A, CG16720), Dop1R1 (Dopamine 1-like receptor 1, CG9652), Dop2R (Dopamine 2-like receptor, CG33517), Act5C (Actin 5C, CG4027). Error bars indicated standard variance of the mean for each gene. Significance was determined for the difference in intensity of the RNAi sample PCR band versus the control w1118 PCR band using a one-sided t-test. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Expression of dsDop1R2 RNAi in the developing fly results in reduced viability, wing malformation and cuticle melanization phenotypes

Dop1R2 RNAi flies that are reared at 29 °C and ubiquitously express the Dop1R2 RNAi construct (Additional file 1: Figure S1) develop normally throughout larval and early pupal stages, but fail to emerge from their pupal cases. When the flies are reared at a lower temperature (i.e., 25 °C), the GAL4/UAS-mediated RNAi gene silencing is attenuated [44] and ‘escaper’ adults emerge (line 1: 19.8 % males and 72.1 % females, line 2: 53.1 % males and 78.1 % females vs. control flies expressing GAL4 alone) (Fig. 3a). The escaper flies display other phenotypes with varying degrees of penetrance, including premature death, abnormal melanization (e.g., abdominal patchiness or complete absence of melanization, Fig. 3b), and/or failure to expand wings (e.g., curly wing, Fig. 3c). As with reduced viability, males show a more pronounced phenotype, with a higher penetrance, than females (data not shown). Two independent Act5C-GAL4 driver lines (FBst0004414 and FBst0003954) resulted in premature death, melanization and wing phenotypes in the progeny.
Fig. 3

Ubiquitous knockdown of Dop1R2 results in reduced adult emergence and wing and/or melanization phenotypes. a Ubiquitous knockdown of Dop1R2 at 29 °C results in 100 % of Dop1R2 RNAi flies (genotype: w1118;UAS-dsDop1R2/+;Act5C-GAL4/+) failing to emerge, compared to control flies (genotype: w1118;UAS-dsDop1R2/+;TM6B/+). At 25 °C, 19.8–72.1 % (line 1) and 53.1–78.1 % (line 2) of Dop1R2 RNAi flies develop into adults (‘escapers’). 25 °C n = 1580 and 29 °C n = 449. Line 1: 4 replicates at 25 °C and 5 replicates 29 °C. Line 2: 3 replicates at 25 °C and 29 °C. Error bars indicate the Standard Error of the Mean (SEM). b Escaper flies may exhibit two other phenotypes: hypomelanization and curly wing. Hypomelanization phenotype appears as reduced melanization of abdominal cuticle (arrows). c Curly wing phenotype appears as bent/curved adult wing (arrows). Driver stock: Act5C-GAL4 (FBst0003954)

The lethal phenotype was recapitulated using two additional UAS-dsDop1R2 lines (Additional file 1: Figure S1 and Additional file 2: Figure S2) generated by the Vienna Drosophila RNAi Center (i.e., 3391-GD and 10524-KK, see Methods). As observed with the Draper/Kopin lab RNAi line, the male escapers (obtained with VDRC driver line 3391-GD) displayed the melanization phenotype (data not shown). Of note, in addition to the above lines, a deletion mutant of Dop1R2 (i.e., damb1) is available that removes part of the 3’coding region of the gene (personal communication, Dr. Han). The resulting transcript would encode a receptor with a truncated intracellular C-terminus. Corresponding flies are viable [4547]. Notably, expression of a truncated GPCR in the context of a deletion that removes the last coding exons is possible, and was previously demonstrated for the Drosophila serotonin receptor d5-TH1A [48].

Analysis of the temporal requirements for Dop1R2 expression suggests a role at the third larval instar and prepupal stage

We have utilized the well-established temperature effect on the GAL4/UAS system (i.e., more efficient at higher 29 °C, vs. lower 25 °C, temperature [44] to probe whether Dop1R2 expression is required during a specific time interval for the flies to complete development. Developing flies were shifted from high (29 °C) to low (25 °C) temperatures, and conversely, during different developmental stages (i.e., embryo, first/second instar larva, third feeding/wandering instar larva, early pupa, late pupa) (Fig. 4a). Regardless of which developmental stage, or direction (high to low vs. low to high), was selected to perform the transfer, flies that were kept at the high temperature throughout the third instar larval stage later arrested at the late pupal/pharate adult stage (Fig. 4b). These experiments indicate that expression of Dop1R2 at the third instar larval stage is critical for survival of the developing progeny (Fig. 4c).
Fig. 4

Down-regulation of Dop1R2 around larval-to-pupal ecdysis leads to developmental arrest. a Schematic of the temperature shift assay. b Analysis of progeny that were switched from 29 °C (high RNAi) to 25 °C (attenuated RNAi) on a defined day post egg laying. n = 1194 (line 1), n = 1107 (line 2). c Analysis of progeny that were switched from 25 °C (attenuated RNAi) to 29 °C (high RNAi) on a defined day post egg laying. n = 1969 (line 1), n = 2212 (line 2). Transfer day and temperature shift, as well as corresponding developmental stage are indicated along the x-axis. Each graph shows the percent of Dop1R2 RNAi (line 1 or line 2) (genotype: w1118;UAS-dsDop1R2/+;Act5C-GAL4/+) that emerge vs. controls (genotype: w1118;UAS-dsDop1R2/+;TM6B/+). Dop1R2 RNAi flies reared at 29 °C fail to emerge as adults. Flies transferred at 25 °C prior to the L3 larval/prepupal stage show higher eclosion. The difference in intervals between (b) and (c) reflects the temperature effect on the length of the life cycle (i.e., the life cycle is shorter when the flies primarily develop at 29 °C, and longer when the flies primarily develop at 25 °C). For each temperature shifts shown in (b) or (c), line 1 and line 2 were each assessed in triplicate. L: larval instar. Error bars indicate the Standard Error of the Mean (SEM)

A preliminary transcriptome analysis of Dop1R2 RNAi flies reveals up-regulation of tyrosine hydroxylase and ecdysone-related genes, as well as stress and immune response genes

Affymetrix GeneChipR Drosophila genome array transcriptome expression analysis was performed in duplicate on early pupal stage Dop1R2 RNAi flies expressing the interference construct ubiquitously under restrictive conditions, and compared to that of corresponding control pupae. Significance was assessed using Genespring array analysis software (Silicon Genetics). A total of 163 genes were identified as significantly differentially expressed following assessment of the two independent transcriptome analyses (Additional file 3: Dataset 1). Among these, only eight genes were down-regulated, with a modest –1.1 to –1.5 fold-difference of expression compared to control flies. Our focus was then shifted to 101 genes that were up-regulated with a fold-difference of  ≥ 1.6 (compared to expression levels in control flies, Fig. 5 and Additional file 3: Dataset 1; arbitrary cutoff of 1.6). Results include a 3-fold increase in expression levels of tyrosine hydroxylase (TH) in Dop1R2 RNAi vs. control flies. The Affymetrix GeneChipR array data discussed in this publication have been deposited into the NCBI's Gene Expression Omnibus (GEO) data repository [49] and are accessible through the GEO Series accession number GSE66496 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66496). FlyBase annotation [50] and DAVID bioinformatic analysis [51] of the genes that are differentially expressed in response to Dop1R2 KD revealed highly significant enrichment (Benjamini corrected p-value range of 4.9E-2 to 4.8E-6) of genes falling under selected ontology (GO) term classes (i.e., heat shock response, immune response, salivary gland development, larval and pupal morphogenesis, Additional file 4: Dataset 2). The related genes that exhibited up-regulation include seven members in the late ecdysone-induced Eig71E (L71) gene family, which were up-regulated ~3-to 6-fold. The expression levels of multiple stress response genes (e.g., Hsp22, Hsp26, Hsp67Bb, Hsp67Bc, Hsp68, Hsp70Bbb, Hsp70Bc, Hsf), antimicrobial peptides/innate response genes (CecA1/A2, dro2/dro3, LysX, IM1, IM2, IM3, IM4, IM10, IM23) associated with gut immune responses [52], and structural components of the cuticle (Cpr72Eb, Cpr65Ec, PCP) also increased in the Dop1R2 RNAi arrested flies compared to controls (Fig. 5 and Additional file 4: Dataset 2). A parallel analysis carried out using WEB-based GEne SeT AnaLysis Toolkit (WEBGestalt) [53, 54] revealed enrichment of genes falling under similar GO term classes (Additional file 5: Figure S3). The relatedness of these groupings is further supported by the many protein-protein interactions revealed using STRING analysis (Additional file 6: Figure S4).
Fig. 5

Transcriptome analysis of Dop1R2 RNAi arrested flies reveals up-regulation of families of related genes. Results indicate an increase in the expression of 101 genes that were significantly up-regulated by ≥ 1.6 times in Dop1R2 RNAi flies (genotype: w1118;UAS-dsDop1R2/+;Act5C-GAL4/+), compared to control flies (genotype: w1118;UAS-dsDop1R2/+;TM6B/+). The fold increase change in transcript level is indicated in parentheses. Statistical significance was determined using a t-test on the average of two independent biological replicates, with a cutoff of p < 0.05. Families were assigned by DAVID functional assignment and by manual annotation using FlyBase. Driver stock: Act5C-GAL4 (FBst0003954)

To further assess results from the microarray analysis, a subset of genes was randomly picked across the main GO categories (Fig. 5). These included Hsp67Bc, Hsp70Bc (heat shock response), Cpr72Eb, Dro2, Dro3, CecA1, LysX (immune response), and Edg91 (ecdysone-dependent genes). Gene expression was assessed by RT-PCR in RNA preparations isolated from independent biological replicates (the corresponding Dop1R2 RNAi and control fly progeny were derived from three novel independent biological replicates (i.e., independent from each other, and from those used for the transcriptome analysis). This analysis confirmed increased transcript levels for all genes assessed [using RNA preparations from three independent Dop1R2 RNAi (and control) biological replicates, Fig. 6]. We also included Rel in the RT-PCR validations (although it fell below the ≥ 1.6-fold cutoff in the microarray analysis) since the corresponding protein is a key effector in the IMD pathway/gut immune response [55]. Using RT-PCR, we observed a slight, but significant, increase in Rel expression, in dsDop1R2 RNAi (vs. control) animals, as was observed by microarray analysis. Quantitative PCR confirmed an increase (4X) in TH transcript levels, in Dop1R2 RNAi flies compared to controls (Additional file 7: Figure S5).
Fig. 6

RT-PCR analysis confirms differential expression of genes in Dop1R2 RNAi flies. a Transcript levels assessed by RT-PCR. RNA obtained from Dop1R2 RNAi (genotype: w1118;UAS-dsDop1R2/+;Act5C-GAL4/+) and control pupae (genotype: w1118;UAS-dsDop1R2/+;TM6B/+) was reverse transcribed, and PCR was performed in triplicate using primer sets corresponding to gene of interest or to Act5C (as a normalization control). b Quantification of transcript levels. The average band intensity of Dop1R2 RNAi PCR products was normalized to control PCR products for Act5C. Error bars indicate the Standard Error of the Mean (SEM), and significance was determined by comparing the difference in intensities of the RNAi PCR bands versus the control PCR bands using an unpaired t-test. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Driver stock: Act5C-GAL4 (FBst0003954)

Analysis of the tissue-specific requirements for Dop1R2 expression suggests a role for receptor function in the salivary gland

To identify the tissue type(s) that underlie the observed phenotypes, Dop1R2 RNAi expression was directed to specific tissues/cell types utilizing a series of GAL4 drivers, and the effects of these genetic manipulations were monitored (Table 1). It is well established that Dop1R2 is abundantly expressed in the mushroom bodies (MB) [20, 56]. However, elav-mediated pan-neuronal expression of the RNAi construct (elav targets all neurons included the MB), and Tab2-mediated expression specifically targeted to the MB failed to compromise viability or to induce gross morphological abnormalities. The vast majority of drivers tested led to progeny with wild-type (WT) phenotypes (Table 1).
Table 1

Effect of tissue-specific down-regulation of Dop1R2. A series of GAL4 drivers was used to down-regulate Dop1R2 expression in specific tissue/cell types. The lethality observed when down-regulating expression ubiquitously was recapitulated only when using the P{GawB}332.2 driver, which expresses GAL4 in the salivary glands and amnioserosa. Semi-lethality was observed when using the P{GawB}17A and P{GawB}c729 drivers. All of the above mentioned drivers resulted in abnormal melanization and cuticle phenotypes

Gal4 driver Symbol

Expression

Phenotype

P{Act5C-GAL4}17bFO1a,b,c

Ubiquitous

Lethal, melanization and wing defect (in escapers)

P{Act5C-GAL4}25FO1a,b,c,d

Ubiquitous

Lethal, melanization and wing defect (in escapers)

P{GawB}332.3a,b,c

Salivary glands, amnioserosa

Lethal, melanization and wing defect (in escapers)

P{GawB}17Aa

Salivary glands, glia, cardia

Semi-lethal

P{GawB}c729a

Salivary glands, female follicle cells, male accessory glands, testis sheath, cyst cells

Semi-lethal, melanization and wing defect (in escapers)

P{GawB}elav[C155]a

Pan-neuronal

WT

P{GawB}Tab2[201Y]a

Primarily in mushroom bodies

WT

P{GawB}c698aa

3IL CNS, not in discs

WT

P{Eip71CD-GAL4.657}TP1-1a

3IL brain and epidermis

WT

Bursicon-α- GAL4a,c

Bursicon-α positive cells

WT

P{Ccap-GAL4.P}16a,c

Crustacean cardioactive peptide-secreting cells

WT

P{GawB}30Aa

Imaginal discs

WT

P{GawB}l(2)T32T32a

Amnioserosa, larval brain, wing discs

WT

P{GawB}c381a

Amnioserosa, embryonic PNS - stage 14

WT

P{Sgs3-GAL4.PD}TP1a

Salivary glands

WT

P{Lsp2-GAL4.H}3a

3IL fat body

WT

P{drm-GAL4.7.1}1.1a

Gastrointestinal tract, malpighian tubules

WT

All phenotypes were assessed on progeny that developed at 29 °C

ain combination with lab generated UAS-dsDop1R2 line 1

bin combination with lab generated UAS-dsDop1R2 line 2

cin combination with UAS-dsDop1R2 VDRC stock FBst0460369

din combination with UAS-dsDop1R2 VDRC stock FBst0477151

WT wild-type phenotype

3IL third instar larva

As a follow-up to this initial study, a more focused selection of candidate drivers was tested, based on the results of the transcriptome analysis. Of particular interest was the Eig71 defensin-like peptides, which are highly expressed in one tissue – the salivary gland – during the L3 wandering/white prepupal stage [57]. The P{GawB}332.3 line (FBst0005398), which expresses GAL4 in the salivary glands, was obtained and utilized to generate salivary gland-expressing Dop1R2 RNAi flies. P{GawB}332.3-directed Dop1R2 knockdown resulted in developmental arrest of the progeny at the pupal/pharate adult stage (Fig. 7a and c), as seen with ubiquitous knockdown of Dop1R2 (Fig. 4b and c). The P{GawB}332.3 knockdown flies also exhibited poorly formed tergites and sternites, with line 1 displaying the most severe phenotype (Fig. 7b). Because P{GawB}332.3 also targets amnioserosal cells, which have a role in germ band retraction and dorsal closure in the developing embryo [58], the fraction of Dop1R2 RNAi embryos hatching into first instar larvae was assessed and compared to that of corresponding control embryos. No evidence of embryonic lethality was found in RNAi-expressing organisms (Additional file 8: Figure S6). In subsequent work, we identified two additional larval salivary gland driver lines (i.e., P{GawB}c729 – FBst0006983), which also targets glia and the proventriculus, and P{GawB}17A – FBst0008474, which also targets female follicle cells, male accessory glands, testis sheath and cyst cells) that induce semi-lethality (72.1 % and 58.2 % lethality, respectively) in the corresponding Dop1R2 RNAi progeny (Fig. 7a and Additional file 9: Figure S7B). Importantly, FBst0005398 and FBst0006983 resulted in progeny displaying wing and/or cuticle abnormalities (Fig. 7b, d and Additional file 9: Figure S7C), as was observed with ubiquitous KD of Dop1R2. For these drivers, the lethal and abnormal morphology phenotypes showed higher penetrance in male flies vs. female flies (data not shown), as was observed with ubiquitous KD of Dop1R2. As indicated in Table 1, one tissue that is common to all three phenotype-positive GAL4 drivers is the salivary gland. Follow-up experiments confirmed GAL4-driven GFP expression in the salivary glands of corresponding larvae (Additional file 10: Figure S8), while other tissues displayed background fluorescence. A fourth salivary gland driver (FBst0006870, for which GAL4 is under the control of the sgs3 gene promoter) did not result in reduced viability (or other phenotypes) in corresponding Dop1R2 RNAi progeny (Table 1).
Fig. 7

Dop1R2 down-regulation using the P{GawB}332.3 driver leads to developmental arrest at the pharate adult stage. Flies that escape lethality display a melanization phenotype. a Expression of Dop1R2 RNAi construct under the control of the P{GawB}332.3 driver (GAL4 expressed in the salivary glands and amnioserosa), induces 98.4 % (line 1) and 99.2 % (line 2) lethality before eclosion. Survival of RNAi flies (genotype: w1118;UAS-dsDop1R2/P{GawB}332.3-GAL4) is expressed as percent control progeny (genotype: w1118;TM6B/GawB-GAL4). n = 208 (line 1), n = 124 (line 2). Crosses with line 1 were done in triplicate; line 2 was used as a confirmatory single experiment. Error bars indicate the Standard Error of the Mean (SEM). b Images of pharate adults dissected out of the pupal case suggest a poorly formed abdomen (arrows, lines 1 and line 2) or incomplete cuticle formation (arrows, line 2). c Analysis of progeny that were switched from 29 °C (high RNAi) to 25 °C (attenuated RNAi) on a defined day post egg laying. Transfer day and temperature shift, as well as corresponding developmental stage are defined along the x-axis. Percent of Dop1R2 RNAi (line 1) (genotype: w1118;UAS-dsDop1R2/P{GawB}332.3-GAL4) that emerge vs. controls (genotype: w1118;TM6B/GawB-GAL4). Dop1R2 RNAi flies reared at 29 °C throughout development fail to emerge as adults, while those transferred to 25 °C prior to L3 larval/prepupal stage show higher eclosion. When flies are transferred between these two temperatures at different stages of development, the time course of lethality is revealed. n = 543. Driver stock: P{GawB}332.3-GAL4 (FBst0005398). d Reduced cuticle melanization (arrows) is observed in dsDop1R2 adult males that escape lethality at 25 °C. L: larval instar

Since Dop1R2 signals though the stimulatory G protein Gαs, we performed a complementary genetic analysis inducing G protein RNAi-mediated knockdown in vivo. Two different UAS-dsGαs (stimulatory G protein) lines, as well as one UAS-dsGαi (inhibitory G protein) line, were used to generate progeny at 29 °C that express dsRNA under control of the P{GawB}332.3-GAL4 driver. Crossing either Gαs RNAi line with the P{GawB}332.3 resulted in pharate adult progeny that failed to eclose, as compared to the corresponding controls. However, the Gαi RNAi progeny develop normally and emerge from their pupal cases as fully formed adults (Fig. 8, Table 2). These findings support the inferences that Gαs signaling in the salivary glands is required for progression to the adult stage, and that the cognate GPCR(s) play an essential role in this tissue/developmental process. In contrast, the inhibitory G protein Gαi does not play a critical role for development in the salivary glands. While this finding does not exclusively pinpoint Dop1R2 as the only essential Gαs-coupled protein in the salivary glands, it supports the premise that we are not targeting Dop2R, which signals via Gαi. As observed with Dop1R2 RNAi, Gαs RNAi under the control of the sgs3 promoter (FBst0006870) does not lead to compromised viability (data not shown). A follow-up molecular analysis confirmed expression of Dop1R2 in salivary glands of wild type prepupae (Fig. 9), as has been documented in other insect species (i.e., cockroach, locust, tick [5964]).
Fig. 8

s-targeted, but not Gαi-targeted KD in the salivary glands results in pre-adult lethality. Expression of either of two Gαs (stimulatory G protein) RNAi constructs [122] under the control of P{GawB}332.3 driver induces lethality before eclosion (line 1 genotype: w1118;P{GawB}332.3-GAL4/+;UAS-dsGαs/+, line 2 genotype: w1118;P{GawB}332.3-GAL4/UAS-dsGαs). Expression of the Gαi (inhibitory G protein) RNAi construct, using the same driver, does not compromise viability (genotype: w1118;P{GawB}332.3-GAL4/+;UAS-dsGαi/+). Survival is expressed as percent of balancer progeny. Driver stocks: Gαs line 1: FBst0455666, Gαs line 2: FBst0477312, Gαi line: FBst0457318. Gαs line 1: n = 141, Gαs line 2: n = 186, Gαi: n = 416. Two replicates were performed for each of the three G protein RNAi driver lines. Error bars indicate the Standard Error of the Mean (SEM)

Table 2

s, but not Gαi, knockdown targeted to salivary glands/amnioserosa leads to developmental arrest

UAS line

Description

Phenotype

UAS-dsGαs (FBst0455666)

s RNAi

Lethal

UAS-dsGαs (FBst0477312)

s RNAi

Lethal

UAS-dsGαi (FBst0457318)

i RNAi

WT

Phenotype assessed on progeny from parental Gal4 driver line P{GawB}332.3 (FBst0005398, salivary glands/amnioserosa, see Table 1) and parental UAS line, as indicated

All phenotypes were assessed on progeny that developed at 29 °C

WT wild-type phenotype

ds double-stranded

Fig. 9

Dop1R2 is expressed in prepupal salivary glands and brain. a RNA obtained from the brain and salivary glands of w1118 prepupae was reverse transcribed, and PCR was performed in triplicate using primer sets corresponding to Dop1R2-RB, Dop1R2-RA/C or Act5C (as a normalization control). Transcript variants A/C and B are detected in the brain, and transcript variant B is detected in the salivary glands (the presence of a low abundance Dop1R2-RA/C transcript in salivary glands cannot be excluded). b Quantification of transcript levels (ImageJ software). Expression is quantified as band intensity for three biological replicates of Dop1R2-RB, or Dop1R2-RA/C, normalized to Act5C

Delivery of a Dop1R2 antagonist to larvae results in reduced viability, abnormal melanization and cuticle defects

Pharmacological assessment of Dop1R2 activity in vitro confirmed that flupenthixol dihydrochloride, with an IC50 of 2.6 × 10−7 M (Additional file 11: Figure S9), is a potent antagonist of this dopamine receptor [24, 59, 65]. Given the in vitro results, this compound was used to manipulate Dop1R2-mediated signaling in vivo, thus providing a means to complement the RNAi genetic manipulations described above. Administering flupenthixol (within a range of 0.25 mM to 4 mM) to Drosophila second instar larvae resulted in a dose-dependent decrease in adult eclosion with an EC50 of 0.8 mM (Additional file 12: Figure S10A) and developmental delay (Additional file 12: Figure S10B). When flupenthixol was administered to Drosophila third instar feeding larvae (at either 5 mM to 10 mM), emerging adults displayed abnormal melanization and cuticle defects (penetrance ~10–13 %, Additional file 12: Figure S10C), and these phenotypes were not observed in corresponding control flies (fed H2O vehicle alone). Importantly, the morphological defects resulting from administration of a Dop1R2 antagonist (as assessed in vitro) are similar to those observed with genetic knockdown of Dop1R2 (Figs. 3b, 7b and Additional file 9: Figure S7C). We cannot exclude that flupenthixol also inhibits signaling at other GPCRs [66]. However, taken together, our complementary pharmacological and genetic data support the premise that Dop1R2 regulates the observed phenotypes.

Discussion

Our understanding of the molecular mechanisms that orchestrate the development of an adult fruit fly continues to expand. Insect metamorphosis relies on temporal and spatial cues that mediate the transition from the larval to the adult stage. Numerous gene families are tightly regulated to ensure normal insect metamorphosis, including genes that trigger larval tissue histolysis and genes that are responsible for protecting the morphing organism against microbial assault, as well as genes that mediate the formation of new adult structures. We show that a Drosophila dopamine receptor, i.e., the D1-like receptor Dop1R2, plays an important role in suppressing the expression of genes, which when up-regulated, lead to developmental arrest.

By using a reverse genetic approach, we show that ubiquitous knockdown of Dop1R2 results in pre-adult lethality that is dependent on receptor function during the third instar larval stage (Figs. 3 and 4). Dop1R2 RNAi adult flies that escape pre-adult lethality display multiple morphological phenotypes including hypomelanization, abnormally shaped/curly wings and defects in the cuticle (in the tergum) (Fig. 3b, c and 7b). The curly wing phenotype displayed by Dop1R2 RNAi escapers is very similar to that seen in flies that overexpress (2-fold increase) tyrosine hydroxylase (TH) in dopaminergic cells [34]. In agreement with this observation, TH is among the genes that respond to reduction in Dop1R2 knockdown (2-4 fold increase in expression levels vs. controls) (Fig. 5, Additional file 7: Figure S5, Additional file 3: Dataset 1). This finding may suggest that: (i) Dop1R2 participates in the negative regulation of TH, or (ii) compensatory mechanisms are triggered to restore normal DA-mediated signaling in the dying Dop1R2 RNAi organisms. The wing and melanization abnormalities seen in Dop1R2 RNAi escapers could be the consequence of TH dysregulation in the epidermal dopaminergic cells of the wing and cuticle. These cells have been shown to synthesize/secrete DA during molting and eclosion in Drosophila [16]. Under normal conditions, a peak of TH activity is detected in late L3 larvae/white prepupae [67, 68], consistent with a role for Dop1R2 during these stages of development.

Decreased Dop1R2 function leads to increased transcription of several cuticular proteins (CPs), including Edg91 and PCP (Figs. 5 and 6, Additional file 3: Dataset 1). Along with ecdysone, many CPs play critical roles in puparial cuticle formation and sclerotization [69]. Proteins encoded by ecdysone-dependent genes (Edg) include temporally regulated CPs that are induced by increased ecdysteroid levels in the hemolymph [69, 70]. In Drosophila, Edg91 responds to 20E pulses and is abundantly expressed in the epidermis during early pupal development, at the time of exocuticle synthesis [71]. ‘Pupal cuticle protein’ (PCP) is also temporally regulated by 20E, and is required for a successful third larval instar to pupal developmental transition. Notably, PCP is most tightly regulated via a small 20E titer rise around the time of head eversion [69, 72]. Dysregulated expression of CPs in Dop1R2 RNAi flies may also contribute to the observed abnormal phenotypes, specifically in the tergum (Fig. 7b).

To better assess the spatial requirements underlying Dop1R2 RNAi-induced developmental arrest in Drosophila, we selectively drove Dop1R2 dsRNA in various tissues/cell types (Table 1). Our microarray analysis, which showed up-regulation of salivary glands specific genes (e.g., the Eig71E genes), suggested involvement of this tissue in mediating Dop1R2 effects. Consistent with this observation, although most tissue-specific drivers resulted in normal progeny, targeting Dop1R2 knockdown to salivary glands (using three different GAL4 drivers, Table 1) led to arrested development/abnormal tergum in corresponding pharate adults. In addition, the corresponding progeny that escaped lethality displayed melanization and/or wing defects that were highly reminiscent of the phenotypes seen following ubiquitous Dop1R2 knockdown (Table 1, Fig. 3). A follow-up molecular analysis confirmed expression of Dop1R2 in salivary glands isolated from wild type prepupae (Fig. 9). This finding correlates with previous studies in other insect species (i.e., cockroach, locust, tick), which have demonstrated dopaminergic innervation of peripheral secretory cells in the acini, and along the ducts, of the salivary glands [6062]. More recently, D1-like dopamine receptors were found in the salivary glands of adult ticks and cockroaches, where they may play a role during the feeding phase, as well as modulate salivary secretion, myoepithelial cell contraction and effects of neuropeptides [59, 62, 63].

A function for Dop1R2 in salivary glands is consistent with: (i) the observed (Dop1R2 RNAi-induced) deregulation of genes that are selectively expressed in this organ (e.g., Eig71E genes), and (ii) the DAVID GO clustering analysis of differentially expressed genes (Figs. 5 and 6, Additional files 3 and 4), which reveals enrichment in salivary gland biological processes. A compelling example comes from the family of Eig71E (aka L71) puff genes that are (concomitantly) induced exclusively in salivary glands, and specifically during puparium formation (they are then repressed ~12 h later) [73]. It is known that the corresponding L71 small defensin-like polypeptides are secreted from the salivary glands between the prepupal cuticle and imaginal epidermis, to help protect the metamorphosing organism against infection [73]. The Eig71E genes participate in the secondary response to 20E (i.e., as “late” genes), which itself depends on the expression of the early-late genes BR-C and E74 [74]. BR-C expression is also up-regulated in Dop1R2 RNAi flies, and derepression of this gene could lead to subsequent induction of the Eig71E genes in Dop1R2 RNAi flies. Our studies support the premise that Dop1R2 acts upstream of selected late genes. Of note, although three out of four salivary gland drivers resulted in corresponding dsDop1R2 progeny displaying comparable phenotypes (Table 1), no phenotype was observed when using the sgs3-GAL4 driver. This may be due to temporal discrepancy between activation of the sgs3 glue gene promoter and that of Dop1R2.

The late pupal death induced by knockdown of Dop1R2 in salivary glands is reminiscent of that observed in flies that down-regulate, in the same tissue, the low abundance ecdysone receptor minor subtype EcR-A [75]. Future studies comparing the levels of EcRs and their subcellular localization in Dop1R2 RNAi flies may prove informative.

The combination of pharmacological and RNAi (i.e., via injected dsRNA) approaches to inhibit/knockdown insect D1-like receptor signaling (in the brain) has provided a powerful means to dissect the role of these receptors in regulating motor behavior [32] or gregarious behavior [33]. Further supporting the role of Dop1R2 in development, pharmacological treatment of larvae with the established D1-like receptor antagonist flupenthixol dihydrochloride [9, 41, 7678] results in pre-adult developmental delay/arrest (Additional file 12: Figure S10A), as well as abnormal melanization and cuticle defects in escapers (Additional file 12: Figure S10C), that recapitulate those observed by genetic manipulation of Dop1R2 expression. Importantly, flupenthixol and other selected compounds that also inhibit the mosquito AaDOP2 receptor, which is the Aedes aegypti ortholog of the fly Dop1R2 receptor, have emerged as promising candidate insecticides to control vector arthropods [9, 66, 79]. Our analysis, which documents drug-induced morphological abnormalities in adults that escape lethality, further highlights the potential of D1-like receptor antagonists as promising insecticides. Such anatomical defects would likely compromise survival of disease-transmitting insect vectors in the field.

Notably, analysis of genes that are differentially expressed in response to reduced levels of Dop1R2 reveals that the vast majority of them (95 %) are up-regulated (Fig. 5 and Additional file 3: Dataset 1). This observation suggests that Dop1R2 may play an important role in repressing gene expression. Functional annotation analysis of the genes for which expression increases ≥ 1.6 times, using DAVID bioinformatic resources [51], identifies enrichment in genes implicated in several biological processes for which temporal regulation is critical (Fig. 5). Several of the gene clusters fall under the GO term categories defense response, immune response, and response to heat, as well as salivary gland morphogenesis and histolysis (Additional file 4: Dataset 2).

Such deregulated activation of the immune system (in response to Dop1R2 knockdown) in the developing fly may contribute to the observed lethal phenotype. It is well-established that in Drosophila the balance between repression and induction of the immune defense is tightly regulated, and ensures optimal growth and size at metamorphosis [8082]. Control of the innate immunity enables larval growth amidst the plethora of bacteria and fungi found in the natural larval feeding environment and ensures high tolerance for the larval gut commensal microbiota, which has been shown to promote development [8385]. Conversely, deregulated immune responses can alter normal fly growth and development. Abdelsadik and Roeder (2010) have demonstrated that chronic activation of the immune system of larval salivary glands is detrimental to fly development and survival [80]. Similarly, Rynes et al. (2012) have shown that chronic inflammation of the larval gut epithelium results in developmental delay, growth retardation and lethality [86].

Recent advances in the field have unraveled an exquisite interplay of negative regulators of the immune deficiency (IMD) pathway that together adapt the immune response to the microbiome encountered by the developing fly (dietary/beneficial or pathogenic). These factors are essential to larval growth and immune homeostasis [55, 81, 8692], and loss-of-function mutations in these negative regulators can result in larval death [86]. Our results suggest that down-regulation of Dop1R2 leads to up-regulation of multiple antimicrobial peptides (AMPs), including the cecropins CecA1 and CecA2 (Figs. 5 and 6), which are gut peptides strongly induced upon infection in an IMD/relish-dependent manner [52, 93]. In non-pathogenic conditions, these AMPs are expressed during metamorphosis [93] and are regulated by ecdysone [94]. Two other AMPs, Dro2 and Dro3, together with LysX, Hsp70Bc, Hsp67Bb and Hsp22 (also on the microarray list), comprise a small group of genes that respond to changes in fly gut microbiota [95]. LysX is a known effector of IMD response [95]. Increased expression of an entire set of Drosophila-specific immune-induced molecules (IMs, i.e., IM1, IM2, IM3, IM4, IM10, IM23, CG18107, CG16836 and IM2-like/CG15065) is observed in Dop1R2 RNAi animals (Fig. 5 and Additional file 3: Dataset 1). These short peptides, which are normally released into the hemolymph following septic injury, are postulated to act as chemokines [96, 97]. Importantly, IM1, IM2, IM3, IM4, IM10, IM23, along with Dro2 and AttB (Fig. 5 and Additional file 3: Dataset 1), were recently identified within a group of 14 AMPs and IMs that are markedly up-regulated in mutant Drosophila deficient in activating transcription factor 3, atf3. Atf3 plays an essential role in larval growth, and is highly expressed in the larval gut, salivary glands and Malpighian tubules [86]. The overlap between the dysregulated gene set (and associated adverse effects on development) induced by Dop1R2 deficiency, and that induced by atf3 deficiency, suggests an important role for Dop1R2 in the control of the immune response.

In addition to antimicrobial peptides, our study shows that the expression levels of multiple heat shock/stress genes increase in response to Dop1R2 deficiency, including the major heat-inducible proteins (Hsp70Bc, Hsp70Bbc, and Hsp68), and small heat shock proteins (Hsp22 Hsp26, Hsp67Bb and Hsp67Bc) (Fig. 5 and Additional file 3: Dataset 1). These chaperones are postulated to play a role in normal development, and under non-heat shock conditions, exhibit a peak of expression during the late L3/early pupal stages [98, 99]. Expression of small hsps is regulated by a rise in the molting hormone ecdysone [100, 101]. Hsp22, Hsp67Bb and Hsp67Bc belong to a group of four hsps that regulate morphogenesis, and buffer developmental processes from environmental assault. Interestingly, the genes that encode Hsp22, Hsp26, Hsp67Bb and Hsp67Bc all cluster within a short (~5.5Kb) genomic region at cytological location 67B on chromosome 3 L (FlyBase, [102]), consistent with possible co-regulation of their expression. High levels of Hsp70 in Drosophila (due to one extra copy of the gene) are sufficient to decrease organismal growth, development and survival to adulthood [103]. Up-regulation of this gene alone in developing Dop1R2 RNAi flies (Figs. 5 and 6) may thus contribute to the observed lethal phenotype that results from reduced Dop1R2 function.

A complementary DAVID GO clustering analysis [51] was used to identify previously published studies with data sets that best correlate with the set of differentially expressed genes in Dop1R2 RNAi flies. Intriguingly, the two most significant reports (i.e., PMID 16990270/Benjamini E-15 and PMID 16264191/Benjamini E-11, respectively, Additional file 4: Dataset 2) both investigate chromatin remodeling and transcriptional activity during metamorphosis [104, 105]. In both studies, the authors show that deficiency in an ecdysone-dependent transcription co-factor affects expression of a limited subset of immune-related genes. The genes identified exhibit substantial overlap with those that respond to Dop1R2 knockdown (Fig. 5, in ecdysone-related and immune diagrams). In support of a potential role of Dop1R2 in the regulation of transcription, sequence analysis (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) reveals the presence of a bipartite nuclear localization signal (the major class of NLS found in nuclear proteins), as well as a BAF1/ABF1 chromatin reorganizing factor motif (http://www.genome.jp/tools/motif/) (Additional file 13: Figure S11), within the Dop1R2 protein. Both features are found nested in the third intracellular loop of the receptor. Interestingly, in mammals selected GPCRs (e.g., adrenergic, catecholaminergic) have been shown to localize at the nuclear membrane where they modulate gene expression [106110]. Notably, Patel et al., have shown that the human orphan GPR158 (a Family C GPCR) harbors a bipartite NLS and translocates to the nucleus where it plays an essential role in modulating cell proliferation [111].

We postulate that under normal conditions, at the time of ecdysone-responsive early gene induction (i.e., during the L3 stage), Dop1R2 in the salivary glands participates in the co-repression of ecdysone-responsive late genes. We propose that the premature release of the Dop1R2 inhibitory effect (using RNAi approaches) translates into increased expression of the L71 defensin-like polypeptides, as well as a series of antimicrobial peptides, stress proteins/chaperones, cuticle and morphogenesis proteins in a de-synchronized manner. This misexpression could be highly detrimental to the developing fly, in agreement with a number of studies discussed above [80, 86, 103].

Conclusions

Taken together, our analyses strongly suggest a role for Dop1R2 in the developmental control of genes at the onset of metamorphosis. Dop1R2 RNAi Drosophila display developmental arrest and morphological defects, as well as show dysregulated expression of genes involved in ecdysone response, morphogenesis and immunity. Tissue-specific RNAi- mediated knockdown of Dop1R2 reveals an important role for this receptor in salivary glands, during the larval-to-pupal ecdysis. In addition, pharmacological inhibition of Dop1R2 (as assessed in vitro) using a D1-like receptor small molecule antagonist recapitulates the abnormal phenotypes observed via genetic manipulation, and highlights the potential of drugs that target Dop1R2 as promising insecticides. Our study provides a framework to further probe the molecular mechanisms that contribute to Dop1R2-induced regulation of fly development.

Methods

Drosophila stocks and culture

Two independent UAS-dsDop1R2 homozygous RNAi stocks (lines 1 and 2) were originally generated at Tufts Medical Center, Boston, MA (Draper/Kopin Laboratory, the lethality phenotype was first documented with these lines). Each line harbors the RNAi transgene on the second chromosome where it is randomly inserted. Line 1 leads to Dop1R2 RNAi flies displaying more severe phenotypes than those obtained with line 2. Although the UAS-transgene expression is under the control of the GAL4/UAS system, subtle differences in expression may occur depending on sequences flanking the site of genomic integration. Two additional UAS-dsDop1R2 stocks (FBst0460369*: w1118;P{GD703}v3391 and FBst0477151: w1118;P{KK110947}VIE-260B) were later obtained from the Vienna Drosophila RNAi Center (VDRC, Vienna, Austria). Two UAS-dsGαs stocks (FBst0455666: w1118;P{GD8547}v24958 and FBst0477312: P{KK107742}VIE-260B) and one UAS-dsGαi stock (FBst0457318: w1118;P{GD12576}v28150/TM3) were also acquired from the VDRC. The w1118 stock and all of GAL4 driver fly lines (with the exception of Bursicon-α-GAL4) were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN): FBst0003954: y1w*;P{Act5C-GAL4}17bFO1/TM6B, Tb1; FBst0004414: y1w*;P{Act5C-GAL4}25FO1/CyO, y+; FBst0000458: P{GawB}elavC155; FBst0004440: w1118;P{GawB}Tab2201Y; FBst0003739: P{GawB}c698a,w1118; FBst0006871: w1118;P{Eip71CD-GAL4.657}TP1-1; FBst0025685: y1w*;P{CCAP-GAL4.P}16; FBst0037534: w*;P{GawB}30A/CyO; FBst0005398: w*;P{GawB}332.3; FBst0008474: w*;P{GawB}17A/CyO; FBst0006983:w*;P{GawB}c729; FBst0006994: w1118;P{GawB}l(2)T32T32/CyO; FBst0003734: w1118;P{GawB}c381; FBst0006870: w1118;P{Sgs3-GAL4.PD}TP1; FBst0006357: y1w1118;P{Lsp2-GAL4.H}3; FBst0007098: w1118;P{drm-GAL4.7.1}1.1/TM3,Sb1, FBst0006874: w*;P{UAS-2xEGFP}AH2. The Bursicon-α-GAL4 stock was generously provided by Dr. W. Honegger (Vanderbilt University, Nashville, TN). All stocks were maintained at 25 °C in a 12 h light:12 h dark cycle on standard Drosophila medium [112]. *FBst0460369 is no longer available at VDRC; however, a corresponding RNAi line using the same RNAi target region is available: FBst0460377, w1118;P{GD703}v3392.

Dop1R2 RNAi construct generation and corresponding UAS-dsDop1R2 transgenic flies

The pUAS-dsDop1R2 RNA interference (‘RNAi’) construct includes the yeast Upstream Activator Sequence (UAS; the binding site for the yeast transcription factor, GAL4) [113], inverted repeats of a 825 bp sequence corresponding to the 3’ coding region of the Dop1R2 receptor cDNA (bp 807–1631 of the Dop1R2 cDNA sequence, with bp 1 corresponding to the start of the translation initiation codon), and a SV40 polyadenylation site. Cloning of the sense and antisense cDNA repeats in the pUAST vector was performed as described previously for a Dop2R RNAi construct [25]. The pUAS-dsDop1R2 RNAi transgene construct (250–300 μg/ml) was coinjected with the P helper plasmid pΠ25.7wc (100 μg/ml) into preblastoderm w1118 Drosophila embryos, according to standard protocols [114]. Multiple independent transformant lines containing the UAS-dsDop1R2 transgene were thus generated at Tufts Medical Center (Draper/Kopin lab) and maintained as homozygotes for the P[UAS-dsDop1R2] transgene. Two lines, designated UAS-dsDop1R2 line 1 and UAS-dsDop1R2 line 2, were used for study.

Generation of Dop1R2 RNAi flies

The interference construct was expressed under the control of the well-characterized GAL4/UAS binary system [113]. UAS-dsDop1R2 homozygous flies, that were either generated in the laboratory or obtained from VDRC (i.e., 3391-GD and 10524-KK, see Methods), were crossed with each of the GAL4 driver lines listed in Table 1. Developing progeny were reared at either 29 °C or 25 °C. Isogenic progeny derived from a cross between the w1118 control strain and the corresponding GAL4 driver line were used as control flies for all molecular and phenotypic analyses.

Phenotypic assessment

Viability, melanization and wing phenotype profiles of the Act5C-GAL4/UAS-dsDop1R2 RNAi progeny were assessed versus those of Act5C-GAL4/w1118 control progeny. To delineate the temporal requirements of Dop1R2 expression for adult eclosion/viability, developing flies were transferred from 25 °C (‘permissive’ condition) to 29 °C (‘restrictive’ condition) during different developmental stages, and emergence was monitored (as a function of developmental stage at transfer). In a complementary analysis, and to assess the spatial requirement of Dop1R2 expression for the organismal viability, Dop1R2 RNAi progeny that express the RNAi construct in specific tissues/cell types were generated at 29 °C, and characterized (the corresponding GAL4 drivers used in the crosses are listed in Table 1).

Assessment of transcript knockdown in Dop1R2 RNAi

RT-PCR analysis was utilized to assess transcript levels in Dop1R2 RNAi flies that express the Dop1R2 RNA interference construct ubiquitously vs. control flies that express the GAL4 transcription factor alone. Analyses were carried out by standard PCR, which provided a cost-effective, yet highly sensitive approach to examine the multiple genes/transcripts under study. Focusing on Dop1R2 transcript levels, we previously observed that results obtained by standard PCR correlated with that obtained by q-PCR (data not shown). RNA was extracted from 10-20 pooled Dop1R2 RNAi early/pale pupae and corresponding control pupae. Total RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY), and purified using the RNeasy Kit with DNase treatment (Qiagen, Valencia, CA), according to the manufacturer’s recommendations. The RNA concentrations were quantified by spectrophotometry. First strand complementary DNA (cDNA) was generated from total RNA (5 ng/μl) using MultiScribe Reverse Transcriptase (Applied Biosystems, Carlsbad, CA). PCR was performed using the GeneAmp PCR core kit (Applied Biosystems, Carlsbad, CA) and the AmpliTaq Gold enzyme (Invitrogen, Grand Island, NY). Amplification was done using the GeneAmp PCR system 9700 thermocycler (Applied Biosystems, Carlsbad, CA). The conditions for PCR included: initial denaturation at 95 °C x 10 min; followed by 32 cycles of amplification: 94 °C x 30 s, 58 °C x 30 s and 72 °C x 1:30 min. The reaction was completed with a seven-minute final extension at 72 °C. The sequences of gene specific primers are provided in Additional file 14: Table S1. Dop1R2 primer pairs were designed to amplify: (i) an amplicon localized within the interference sequence (i.e., both forward and reverse primers anneal within RNAi sequence – “in/in pair”) to confirm expression of the RNAi repeats, as well as (ii) an amplicon that corresponds to a region of Dop1R2 mRNA within and outside the RNAi sequence (i.e., the forward anneals within RNAi sequence and the reverse anneal outside) – “in/out pair”) enabling assessment of endogenous Dop1R2 mRNA levels. To assess whether the RNAi construct exerted non-specific off-target effects, primer pairs corresponding to other biogenic amine receptors [i.e., Oamb (CG3856), Oct-TyrR (CG7485), 5-HT1A (CG16720), Dop1R1 (CG9652), Dop2R (CG33517)] were designed, so that the sequence with the most extensive homology (as assessed by NCBI BLAST analysis) between these transcripts and the Dop1R2 sequence was amplified for each, respectively. The primers were synthesized at the Tufts University Molecular Core (Tufts University, Boston, MA) and are listed in Additional file 14: Table S1. PCR products were run on a 1 % agarose gel with ethidium bromide, and photographed using a Multi Image Light Cabinet and camera (Alpha Innotech Corporation, San Leandro, CA). Alphaimager 2200 v5.04 imaging software (Alpha Innotech Corporation, San Leandro, CA) was used to visualize the bands and determine band intensity and saturation point. RT-PCR analysis was performed in triplicate using independent biological replicates. For each GPCR gene/transcript assessed, the difference in the PCR signal intensities in Dop1R2 RNAi and control w1118 flies were obtained and significance evaluated using a pooled variance t-test.

Transcriptome analysis and RT-PCR validation

Gene expression analysis was performed on the GeneChipR Drosophila genome array (DrosGenome1) using Affymetrix Gene Array technology, according to standard Affymetrix protocols (http://www.affymetrix.com/support/technical/byproduct.affx?product=fly). Total early pupal RNA was isolated and purified as described in ‘Assessment of transcript levels’ above, and double-strand cDNA was obtained using SuperScript Double Stranded cDNA Synthesis kit (Invitrogen, Grand Island, NY). In vitro transcription and RNA labeling was performed using Enzo BioArray High Yield RNA transcript (Affymetrix, Santa Clara, CA), according to the manufacturer recommendations. Data were analyzed using the Microarray Suite program (Affymetrix, Santa Clara, CA), as well as Genespring array analysis software (Silicon Genetics). Only genes with expression signal called as “M” (marginal present) or “P” (present) in both replicates were selected for further statically analysis. A t-test was performed to assess the significance of differential expression between the transgenic RNAi lines and the controls. Only genes that exhibited significant differences (p <0.05) in expression levels compared to controls in both experiments were considered for further bioinformatic analysis using DAVID (see following ‘Bioinformatic analysis’). The complete analysis is provided in Additional file 3: Dataset 1. RT-PCR analysis was used to further assess/validate selected differentially expressed genes. Early pupae were collected, and total RNA was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO) according to manufacturer’s instructions. Synthesis of first strand cDNA was performed using 25 ng/μl total RNA and MMuLV Reverse Transcriptase (Invitrogen, Grand Island, NY). All primers were designed to span exon boundaries (except in circumstances of single-exon transcripts), to avoid gDNA amplification (primer sequences are provided in Additional file 14: Table S1). The conditions utilized for RT-PCR were: 95 °C for 2 min, followed by 30 cycles of 95 °C × 15 s, 50-55 °C × 30 s, 68 °C × 10 s, and completed with one cycle of 72 °C × 10 min. Samples were run on a 2 % agarose gel with ethidium bromide and imaged to measure band intensity using ImageJ software (NIH, Bethesda, MD). The PCR products were confirmed by sequencing (Eton Bioscience Inc., Boston, MA) the corresponding amplicon excised from the gel. For quantitative RT-PCR, SYBR Green fluorescence using the Quantitect SYBR Green kit (Qiagen, Valencia, CA) was used to quantify production of a PCR-generated cDNA fragment (primers sequences are listed in Additional file 14: Table S1). Amplification and data analysis were performed using the ABI Prism 7700 (Applied Biosystems, Carlsbad, CA). The PCR conditions utilized for RT-PCR were: 50 °C × 2 min, 95 °C × 15 min, followed by 40 cycles of 95 °C × 15 s, 64 °C × 45 s.

Bioinformatic analysis

All of the identified differentially expressed genes were used for functional annotation analysis with the DAVID Bioinformatics Resource 6.7 [51]. Using the functional annotation tool for Drosophila melanogaster, a total of 101 genes that were up-regulated by ≥ 1.6-fold were analyzed for GO class and pathway associations. For any identified gene ontology (GO) term and pathway, enrichment was considered significant if the p-value observed was < 0.05 [115]. Alternatively, the set of genes was analyzed using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt), designed for functional genomic, proteomic and large-scale genetic studies. The program uses the hypergeometric test for enrichment evaluation analysis, and Benjamini-Hochberg multiple test adjustment, to assess enrichment significance [54, 116]. In addition, protein-protein interaction analysis was performed with STRING 9.1 for all of the genes up-regulated by ≥ 1.6-fold [117].

Analysis of Dop1R2 expression in prepupal tissues

The brain and salivary glands were dissected from prepupal w1118 D. melanogaster. For each tissue, total RNA was isolated, and cDNA was prepared using 50 ng/μl total RNA (as detailed in ‘RT-PCR validations’, above). Dop1R2 was amplified using primers that span nucleotide positions 1521-1637 (isoforms A and C) or 1598-1711 (isoform B), with bp 1 corresponding to the start of the translation initiation codon. As an endogenous control, Act5C (CG4027) was amplified and used for normalization. To enable detection of tissue contamination, primers were designed to amplify repo (CG31240) cDNA (repo expression is enriched in glia) to provide a brain-specific probe [118]), and sgs5 (CG7596) cDNA, to provide a salivary gland-specific probe. All primers were designed to span exon boundaries (sequences are listed in Additional file 14: Table S1) to avoid gDNA amplification. PCR conditions and imaging were performed as mentioned in the validation portion of ‘Transcriptome analysis and RT-PCR validation.’ To confirm that the amplicon corresponded to Dop1R2, DNA bands were excised from the gel and sequenced.

In vitro Dop1R2 pharmacology

Luciferase assays were performed as previously described, with minor modifications [119]. HEK293 cells in 96-well plates were grown in serum-free Dulbecco’s modified eagle medium with antibiotics. After 48 h, cells were transfected using PEI (1 μg/ml) with the following constructs: the Drosophila Dopamine 1 receptor 2 (Dop1R2) cloned into pcDNA1.1 (4 ng/well), a CRE-LUC-HCL-PEST luciferase reporter gene (5 ng/well), and β-galactosidase-encoding plasmid (5 ng/well) as a transfection control. For agonist assays, cells were treated with the indicated concentrations of dopamine hydrochloride 24 h after transfection (Product H8502, Sigma, Natick, MA). For antagonist assays, butaclamol hydrochloride (Product D033, Sigma, Natick, MA) or flupenthixol dihydrochloride (Product 4057, Tocris Bioscience, Bristol, UK) was added to cells for 15 min prior to the addition of 1 μM dopamine. For both agonist and antagonist assays, cells were treated with compound for 4 h at 37 °C. Luciferase activity was quantified as an index of Dop1R2 signaling. Activity data were normalized relative to β-galactosidase activity as a control for transfection efficiency.

In vivo treatment of larvae with a Dop1R2 small molecule antagonist

Adult w1118 D. melanogaster were allowed to mate for 12 h at 25 °C to obtain developmentally synchronized eggs laid on Drosophila medium. All adults were removed, and larval development was allowed to continue for ~48 or ~72 h, to obtain L2 or L3 instar larvae. Flupenthixol dihydrochloride (Product 4057, Tocris Bioscience, Bristol, UK) was prepared as a 25 mM stock solution in dH2O. L3 larvae were fed flupenthixol at 5 mM or 10 mM. The drug solutions, or dH2O vehicle-only, were used to prepare instant fly food (Carolina Biological Supply Company, Burlington, NC) as follows: 0.5 g of fly food was placed into 25 x 95 mm polystyrene tubes (Dot Scientific Inc., Burton, MI) with 2 ml of solution (prepared in dH2O and 0.1 % (v/v) including Fast Green Fast Green FCF dye (Product F7258, Sigma-Aldrich, St. Louis, MO). Fifty L2 or thirty L3 instar larvae were inserted gently into tubes that were kept in a humid chamber at 25 °C during the course of the treatment.

Ethics approval and consent to participate

The use of Drosophila melanogaster for research purposes does not require ethical approval.

Consent for publication

Not applicable.

Availability of data and material

The data sets supporting the results of this article are available in the NCBI's Gene Expression Omnibus data repository, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66496.

Declarations

Acknowledgements

The authors thank the Bloomington Drosophila Stock Center (NIH P40OD018537), VDRC [120] and H.W. Honegger for fly strains. We also thank Dr. Jamie Doyle for valuable input and suggestions pertaining to this manuscript, and Ms. Ci Chen for informed technical advice.

Funding

This research was funded by NIH/NIDDK grant number R01DK070155-04 (Alan S. Kopin) and by the Biology Department at Boston College. The Boston College University Libraries, in collaboration with the Provost, have generously provided funding for the open access publication costs.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biology, Boston College
(2)
Molecular Cardiology Research Institute, Tufts Medical Center
(3)
Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University
(4)
Discovery Research, Biogen Idec
(5)
Present Address: Department of Biology, Stanford University

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