- Email: [email protected]
Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster
Peptides 68 (2015) 50–57
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster Ji Chen a , Min Sung Choi a , Akira Mizoguchi b , Jan A. Veenstra c , KyeongJin Kang d , Young-Joon Kim e,∗ , Jae Young Kwon a,∗∗ a
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan c Université de Bordeaux, INCIA UMR 5287 CNRS, 33405 Talence, France d Samsung Biomedical Research Institute, Department of Anatomy and Cell Biology, School of Medicine, Sungkyunkwan University, Suwon 440-746, Republic of Korea e School of Life Sciences, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 4 January 2015 Accepted 7 January 2015 Available online 16 January 2015 Keywords: Orcokinin Neuropeptide Drosophila melanogaster
a b s t r a c t Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1–5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model. © 2015 Elsevier Inc. All rights reserved.
Introduction Neuropeptides are an evolutionarily ancient class of signaling molecules that function as neurohormones, neuromodulators, and neurotransmitters, and regulate various developmental and physiological processes in almost all animal species [17,30]. The fruit fly Drosophila melanogaster genome has at least 45 neuropeptide genes, which encode much larger numbers of mature
∗ Corresponding author at: School of Life Sciences, GIST (Gwangju Institute of Science and Technology), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea. Tel.: +82 62 970 2492. ∗∗ Corresponding author at: Department of Biological Sciences, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea. Tel.: +82 31 299 4495. E-mail addresses: [email protected] (Y.-J. Kim), [email protected] (J.Y. Kwon). http://dx.doi.org/10.1016/j.peptides.2015.01.002 0196-9781/© 2015 Elsevier Inc. All rights reserved.
neuropeptides [6,13,17]. The number of genes encoding neuropeptides continues to increase as additional genes and mRNA species from animal genomes are uncovered thanks to advances in bioinformatical tools and RNA sequencing technology. Like other eukaryotic genes, a neuropeptide gene can produce multiple splicing variants, and alternative mRNA splicing further increases the repertoire of mature neuropeptides. Orcokinins were first identified as a myostimulatory factor in the hindgut of the crayfish Orconectes limosus [28]. Classical biochemical isolation, mass spectrometry, and most recently bioinformatic approaches have subsequently identified orcokinin-like peptides in numerous insect species [2,3,7,10,11,14,16,18,22,25]. The physiological function of orcokinins has been studied in several insect species. Orcokinins appear to have a role in the circadian clock controlling locomotory activity in the cockroach Leucophaea maderae [8], and act as neuronal prothoracicotropic factors in Bombyx mori [32]. A recent study revealed that the orcokinin
J. Chen et al. / Peptides 68 (2015) 50–57
gene produces at least two alternatively spliced mRNA transcripts that encode specific peptides in major orders of insect species, including Diptera (Anopheles gambiae), Lepitoptera (Bombyx mori), Hymenoptera (Apis mellifera), and Hemiptera (Rhodnius prolixus) [22,29,32]. According to these analyses [29,32], orcokinin A appears to occur mainly in the central nervous system (CNS), and orcokinin B in the CNS and anterior midgut. Two alternatively spliced forms of orcokinin were also shown to exist in D. melanogaster [31]. This study revealed that an antiserum against the predicted mature orcokinin B peptide showed immunoreactivity in the midgut of both larvae and adults. However, the neuronal expression patterns of the two orcokinin isoforms in Drosophila remain unknown. Here, we characterize the expression pattern of the two orcokinin isoforms in Drosophila using isoform-specific mRNA and protein staining, and transgenic orcokinin-GAL4 flies. The differential expression patterns of the two alternative mRNA splicing variants of the orcokinin gene suggest that the two isoforms have likely evolved to mediate different physiological functions. Materials and methods Drosophila stocks Flies were grown on standard cornmeal/agar culture medium at an average culture temperature of 23 ◦ C. To construct orcokininGAL4 transgenic flies, the orcokinin-GAL4 transgene was first prepared by cloning the 5 upstream region of orcokinin (−504 to +26; +1 indicates the transcription start site) amplified with the forward primer (5 -tatagcggccgcgcttgcagatgaatgggatt-3 ) and reverse primer (5 -gcgcgctctagaaccgccaggagcacatatag-3 ) into the pAGAL4 vector. This transgene was then inserted into a specific site on the second chromosome (VIE-72A, a gift from B.J. Dickson) using the C31 system [5]. pAGAL4 was prepared by inserting a sitespecific integration site (attB) into the 7–74 site of the pPTGAL4(+) vector [26]. For in situ hybridization and immunostaining, w1118 and orcokinin-GAL4; UAS-mCD8-GFP flies were used. UAS-mCD8GFP [15] and pdf-GAL4 [24] were described previously. Due to no obvious gender-specific differences in orcokinin expression (data not shown), we present data obtained from males. In situ hybridization The CNS and intestinal tissues from 3rd instar larvae or 5 to 9 day-old adults were dissected in phosphate-buffered saline with 0.2% Triton X-100 (PBS-T). For the adult intestines, whole abdomens with incisions were fixed and subjected to subsequent staining steps, and the intestine was dissected out for mounting. Samples were fixed in 4% paraformaldehyde in PBS-T for 2–4 h at room temperature, and washed with PBS-T. Proteinase K (50 g/ml in PBS-T) treatment was performed for 10 min, and then stopped by adding glycine-PBS-T (2 mg glycine/ml PBST). After washing with PBS-T, the tissues were postfixed in 4% paraformaldehyde in PBS-T for 1 h and washed with PBS-T. After prehybridization in hybridization solution (50% formamide, 50 g/ml heparin, 100 g/ml salmon testes DNA, 5× SSC, 0.1% Tween 20) for 10–20 min at 48 ◦ C, the dissected tissues were incubated with the ssDNA probe in hybridization solution for at least 20 h at 48 ◦ C. Digoxygenin (DIG)-labeled ssDNA probes targeting exons specific to each orcokinin isoform were prepared using the PCR-DIG Probe Synthesis kit (Roche). The following PCR primers were used to generate probes targeting the isoform-specific exons: orcokinin A-specific exon (+116 to +384; forward primer 5 -attgatttcggctgccaatggac-3 , reverse primer 5 ctagacgagctggttgaggatgc-3 ), and orcokinin B-specific exon (+153 to +402; forward primer 5 -atctgccagcgttaagagaattg-3 , reverse
51
primer, 5 -ctagtcgcttaggaactgtctac-3 ). The probe was mixed into hybridization solution (10 l of probe in 90 l of hybridization solution) and boiled for 40–45 min just before hybridization. After hybridization, the dissected tissues were washed with hybridization solution for 2–4 h at 48 ◦ C, and briefly washed with 1:1 hybridization solution: PBS-T and PBS-T only. The samples were then blocked with 3% goat serum in PBS-T for 30 min at room temperature. Two methods were used for amplification of the digoxygenin signal. The first method used alkaline phosphatase-labeled sheep anti-digoxygenin antibody (1:1000, Roche, Mannheim, Germany) to amplify the digoxygenin signal overnight at 4 ◦ C. Tissues were subsequently stained with nitro blue tetrazolium and 5-bromo4-chloro-3-indolyl-phosphate solution (NBT-BCIP, Roche) diluted 1:50 in alkaline phosphatase buffer (100 mM Tris, 50 mM MgCl2 , 100 mM NaCl, 0.1% Tween 20, pH 9.5). The second method utilized fluorophore-conjugated tyramide to amplify the digoxygenin signal. A tyramide signal amplification kit (Molecular Probes, Cat. No. T20924) was used following the manufacturer’s instructions. Briefly, rabbit monoclonal digoxigenin antibody (1:1000, Invitrogen, Cat. No. 700772) was added and the samples incubated overnight at 4 ◦ C. Samples were washed three times with PBS-T and 3% goat serum in PBS-T 30 min, before adding the goat antirabbit HRP-conjugated secondary antibody (1:200) for incubation at room temperature for at least 1 h. After three washes with PBS-T, Alexa Fluor® 568-labeled tyramide was added for 30 min at room temperature. Samples were then washed and mounted on a slide. The tyramide signal amplification method has the advantage of enabling double labeling of the sample with another antibody and simultaneous detection of fluorescent signals. When double labeling with orcokinin antibodies was performed, primary antibodies were added after the steps described above, and subsequent steps followed the immunostaining protocol described below. Immunostaining For the immunohistochemistry of intestinal cells, whole abdomens were stained and the stained intestines were dissected out for mounting before imaging. A previous immunocytochemistry protocol was followed [20], with minor modifications. Briefly, whole abdomens were dissected in phosphate buffered saline with 0.2% Triton X-100 (PBS-T, pH7.2), and fixed in 4% paraformaldehyde in PBS-T buffer for 2 h on ice. After 3 washes with PBS-T, samples were blocked with 3% goat serum in PBS-T for at least 30 min. Abdomens were incubated with primary antibodies or antiserum for 1–2 days at 4 ◦ C, washed with PBS-T, and incubated with secondary antibodies overnight at 4 ◦ C. For the immunohistochemistry of the brain and ventral nerve cord (VNC), adult brains and VNC were dissected and prepared as described [21,23]. Samples were incubated with primary antibodies or antiserum for 48 h at 4 ◦ C, washed with PBS-T, and incubated with secondary antibodies overnight at 4 ◦ C. The primary antibodies or antiserum used were rabbit antiGFP (1:1000, Molecular Probes), mouse anti-Bombyx orcokinin A (1:1000 [32]), mouse anti-Drosophila orcokinin B (1:1000 [31]), rabbit anti-leucokinin (1:200 [1]), and mouse anti-Prospero (1:10, Developmental Studies Hybridoma Bank at the University of Iowa). Prospero is a marker for enteroendocrine cells. The secondary antibodies used were goat anti-mouse and goat anti-rabbit IgG conjugated to either Alexa Fluor® 568 or Alexa Fluor® 488 (1:1000, Molecular Probes). Imaging and microscopy A Zeiss LSM 510, 700 laser-scanning confocal microscope was used to image fluorescent samples, and a Leica DM2500 microscope
52
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 1. Drosophila orcokinin gene structure. The Drosophila genome database (http://www.flybase.org) predicts different mRNA variants (orcokinin-RA and -RC), each of which encodes either of two mature orcokinin isoforms. Gray and red colored boxes indicate the untranslated and translated regions of orcokinin transcripts, respectively. The genomic fragment used to generate orcokinin-GAL4 is indicated by a black box. The regions that correspond to fully processed mature orcokinin isoforms are marked as thick black lines below the exon. The single-letter amino acid sequence of each mature isoform is shown in parentheses. Isoform-specific in situ probes were prepared from isoform-specific exons indicated by a blue (isoform A) or green box (isoform B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
with a digital camera (Canon EOS 700D) was used to image in situ hybridized samples. Results Two isoforms of orcokinin are predicted in the Drosophila genome According to the most recent version of the Drosophila genome database (FlyBase Release 6), the Drosophila orcokinin gene is predicted to produce mRNA splicing variants, each of which produces either of two mature orcokinin isoforms with distinctly different amino acid sequences (Fig. 1). Since studies in other insects suggested that orcokinin A and orcokinin B have distinct expression patterns in the CNS and intestine [29], we characterized the expression of both Drosophila isoforms in those tissues. For this purpose, we examined the expression pattern of an orcokinin-GAL4 driver, as well as endogenous expression of orcokinin A and orcokinin B using in situ hybridization and isoform-specific antisera, in the CNS and intestine. Transcripts specific for Drosophila orcokinin A and B share a common 5 upstream region, and the entire 5 region upstream of the translation initiation codon of the orcokinin gene was combined with the GAL4 open reading frame to construct the orcokinin-GAL4 driver (black box in Fig. 1). Exons specific to each isoform were amplified to prepare probes for in situ hybridization (green and blue boxes in Fig. 1). Orcokinin A is expressed in the CNS, but not in the intestine A digoxygenin-labeled ssDNA probe targeting the orcokinin A-specific exon was used to examine orcokinin A mRNA expression in the adult CNS. In situ hybridization in the adult fly brain labeled one pair of cell bodies near the center of the brain and four additional pairs near the optic lobes (Fig. 2A). The cell bodies located near the center of the brain were much larger in size, and appear to be located in the posterior region of ventrolateral neuropils, based on comparison of DIC images with 3D stacks of the adult brain provided by Virtual Fly Brain (http://www.virtualflybrain.org/site/stacks/index.htm [12]) (Fig. 2A). Thus, we named the larger cells orcoA-PLP (orcokinin Apositive posteriorlateral protocerebrum neurons). The four pairs of cells occurring laterally in the brain near the accessory medulla are closely positioned to the LNv cells which express pdf-GAL4, but are not the LNv cells per se (Fig. 2F and G). Thus, we named the four small neurons orcoA-AME (orcokinin A-positive accessory medulla neurons). The same in situ labeling was observed in
the adult brain regardless of whether alkaline phosphatase or a fluorophore-conjugated tyramide was used to amplify the digoxygenin signal (Fig. 2B). Drosophila orcokinin A expression examined by an antiserum against Bombyx orcokinin A showed an expression pattern identical to in situ mRNA staining (Fig. 2C). GFP expression driven by the orcokinin-GAL4 transgene was broader than the expression observed by in situ hybridization or antibody staining (Fig. 2D), likely reflecting some ectopic expression due to limitations of the GAL4-UAS system. Nevertheless, all adult brain cells positive for anti-orcokinin A staining overlapped with orcokininGAL4 transgene-expressing cells (Fig. 2D and E). Two pairs of cell bodies are clearly labeled in the adult ventral nerve cord (VNC) by both in situ hybridization and anti-orcokinin A staining (Fig. 3A and B), and all of these cells overlap with orcokininGAL4 transgene-expressing cells (Fig. 3C). Ectopic expression of the orcokinin-GAL4 transgene is also observed in the VNC (Fig. 3C). The orcokinin A-expressing cell bodies are located dorsally between the metathoracic neuromeres and abdominal ganglion, and were named orcoA-VNC. In the larval central nervous system (CNS), five pairs of abdominal neurons (orcokinin A-positive lateral abdominal neurons, or orcoA-LA) were specifically labeled by in situ hybridization specific for orcokinin A mRNA (Fig. 4A), anti-orcokinin A antibody staining (Fig. 4B), and orcokinin-GAL4 transgene expression (Fig. 4C). Each pair of orcoA-LA occurs laterally in each of the 5 abdominal neuromeres, reminiscent of the expression pattern of another neuropeptide drosokinin (aka Drosophila leucokinin), which also occurs in laterally paired neurons in the abdominal neuromeres A1–A7 [19]. Thus, we compared orcokinin A and drosokinin-expressing cells by double antibody staining, and found that the orcoA-LA neurons in the abdominal neuromeres are juxtaposed to those expressing drosokinin (Fig. 4D). In contrast to the expression in the CNS, larval and adult intestines are completely devoid of orcokinin A expression when examined with in situ hybridization and antibody staining (data not shown). Orcokinin B occurs mainly in the midgut enteroendocrine cells In situ hybridization of an orcokinin B-specific exon failed to detect orcokinin B mRNA-positive neurons in the adult CNS, except for one unpaired large cell in the abdominal ganglion (orcokinin Bpositive unpaired AG neuron or orcoB-uAG) (Fig. 5A). Likewise, the orcoB-uAG cell is the only cell in the CNS labeled by anti-orcokinin B staining (Fig. 5B). Cells expressing the orcokinin-GAL4 transgene also included orcoB-uAG, despite some ectopic expression in the abdominal ganglion (Fig. 5C). Unlike the A-isoform, the B-isoform
J. Chen et al. / Peptides 68 (2015) 50–57
53
Fig. 2. Drosophila orcokinin A expression in the adult brain. (A) In situ hybridization to orcokinin A shows expression in one pair of cell bodies near the center of the brain (orcoA-PLP) and 4 pairs of cell bodies in the accessory medulla (orcoA-AME). A, A , A are all images of the same adult fly brain. A is at an optic plane more posterior than A . A is a magnified image of the white box in A . The arrowheads in A indicate the cell bodies of four neurons expressing orcokinin A in the accessory medulla. (B) Fluorescence in situ hybridization, (C) orcokinin A antibody staining, and (D) orcokinin-GAL4 driven GFP reporter expression confirm the adult brain expression pattern observed in A. (D) All cells detected in the adult brain by orcokinin A antibody staining overlap with orcokinin-GAL4 transgene-expressing cells. (E) shows magnified images of the white boxes in D. (F) The 4 pairs of cells expressing orcokinin A in the accessory medulla are different cells from the LNv cells which express the pdf-GAL4 reporter. (G) is a magnified image of the white box in F. All images are from 5 to 9 day-old adult males. Dorsal is to the top. The scale bar indicates 50 m in A, A , B, C, D and F, and 100 m in A , E and G.
was not detected in the larval CNS by in situ hybridization (data not shown). In contrast to orcokinin A, we observed that orcokinin B is expressed in both adult and larval intestines by in situ hybridization (Fig. 6A, data not shown). This is consistent with a previous report that showed the presence of orcokinin B immunoreactivity in intestinal endocrine cells [31]. In situ hybridization also revealed that orcokinin B is mainly expressed in the adult anterior and
middle midgut, in cells whose morphology and position are characteristic of enteroendocrine cells (Fig. 6A). Intestinal cells detected by orcokinin B antisera are also labeled by the orcokinin-GAL4 transgene, although some intestinal cells positive for orcokinin-GAL4 lack anti-orcokinin B immunoreactivity (Fig. 6B). Colocalization of Prospero, an enteroendocrine marker, and orcokinin-GAL4 provides further verification that orcokinin B is expressed in the enteroendocrine cells (Fig. 6C).
54
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 3. Drosophila orcokinin A expression in the adult ventral nerve cord. (A) Two pairs of cell bodies are labeled in the adult ventral nerve cord by in situ hybridization. (B) Orcokinin A antibody staining and fluorescence in situ hybridization label the same cells in the adult ventral nerve cord. (C) Orcokinin A antibody staining and orcokinin-GAL4 driven GFP reporter expression label the same cells (orcoA-VNC, arrowheads) in the adult ventral nerve cord. Anterior is to the top. All images are from 5 to 9 day-old adult males. All scale bars indicate 50 m.
Fig. 4. Drosophila orcokinin A expression in the larva CNS. (A) In situ hybridization to orcokinin A labeled 5 pairs of neurons in the larval CNS. A is a magnified image of the white box in A. Double labeling of orcokinin A by (B) antibody staining and fluorescence in situ hybridization, and (C) antibody staining and orcokinin-GAL4 driven GFP reporter expression confirm the expression in five pairs of cells. (D) Orcokinin A-expressing cells are different cells from drosokinin-expressing cells. All images are from 3rd instar larvae. All scale bars indicate 50 m.
J. Chen et al. / Peptides 68 (2015) 50–57
55
Fig. 5. Drosophila orcokinin B expression in the adult CNS. (A) In situ hybridization of orcokinin B showed expression in a single unpaired cell in the abdominal ganglion. A is a magnified image of the white box in A. (B) Double labeling of orcokinin B by antibody staining and fluorescence in situ hybridization confirm the expression in one cell in the abdominal ganglion. B is a magnified image of the white box in B. (C) Double labeling of orcokinin B by antibody staining and orcokinin-GAL4 driven GFP reporter expression confirm the expression in one unpaired cell in the abdominal ganglion. Arrowheads indicate the single cell co-expressing orcokinin B antibody staining and orcokinin-GAL4 driven GFP reporter expression (orcoB-uAG). Anterior is to the top. All images are from 5 to 9 day-old adult males. All scale bars indicate 50 m.
Discussion Here, we examine the mRNA and peptide expression of the two orcokinin isoforms in the Drosophila CNS and intestine by in situ hybridization and immunolabeling. Our analysis demonstrates that the two isoforms display specific and distinct expression patterns in the CNS and midgut enteroendocrine cells: orcokinin A is expressed in the CNS, and orcokinin B is mainly expressed in the midgut. We also establish and confirm that the expression of an orcokininGAL4 driver recapitulates that of orcokinin peptides and mRNAs in the CNS and enteroendocrine cells, providing a useful genetic tool to manipulate the activities of orcokinin-producing cells in future functional analyses. In a study of Bombyx orcokinin expression, orcokinin mRNA was observed in the nervous system and midgut enteroendocrine cells, whereas orcokinin immunoreactivity was detected only in the nervous system [32]. Since the in situ probe used in the Bombyx study was specific to exons common to both the orcokinin A and B isoforms, the in situ results likely reflect a composite of both A and B isoform RNA expression. Considering that the Bombyx orcokinin antibody was raised against an orcokinin A-specific region, the previous Bombyx study is consistent with the conclusions we made in this study: the A- and B-isoforms of orcokinin mainly occur in
the CNS and in the midgut, respectively. In Rhodnius prolixus, the orcokinin A isoform is expressed in the CNS, and the B isoform in the CNS as well as the anterior midgut [29]. In Drosophila, orcokinin B was reported to be expressed in the midgut of adults and larvae [31]. Together with this previous report, our results provide further support that the two alternative forms of orcokinin have evolved to function separately in two different tissues, the nervous system and the intestinal enteroendocrine system, across different insect species. Based on mRNA and peptide expression patterns, we can hypothesize that the Drosophila orcokinin A isoform mainly functions in the nervous system. Some clues to the role of orcokinin A in the accessory medulla of Drosophila can be inferred from studies in other insects. In the cockroach Leucophaea maderae, about 30 neurons in the accessory medulla are labeled by orcokinin immunostaining [9,27]. Four groups of neurons (MCI, MCII, MCIII, and MCIV) in the cockroach accessory medulla have been proposed to act in coupling the two bilaterally symmetric circadian pacemakers located in the bilateral accessory medullae [27]. PDFand orcokinin-reactive PDFMe neurons in the MCI group were proposed to transmit circadian phase information for coupling, and orcokinin-immunoreactive VMNe neurons in the MCII group were proposed to transmit light information to the circadian
56
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 6. Drosophila orcokinin B expression in the adult intestine. (A) In situ hybridization showed that orcokinin B is expressed in the adult anterior midgut, in cells whose morphology and position are characteristic of enteroendocrine cells. A and A are magnified images of the white box in A, and show images of different focal planes. The cells in A marked by arrowheads have a shape characteristic of enteroendocrine cells. (B) Double labeling of orcokinin B antibody and orcokinin-GAL4 driven GFP reporter expression confirm expression in the anterior midgut. (C) Cells labeled by orcokinin-GAL4 driven GFP reporter expression overlap with cells expressing Prospero, an enteroendocrine cell marker. All images are from 5 to 9 day-old adult males. The scale bar indicates 100 m in A and C, and 50 m in A , A and B.
clock [8,27]. Orcokinin injections into the vicinity of the accessory medulla caused phase shifts in circadian locomotor activity similar to phase shifts caused by light pulses, leading to the suggestion that orcokinin-related peptides play an important role in light entrainment of the circadian clock [8]. It thus appears to be a viable hypothesis that the Drosophila orcokinin A form-immunoreactive neurons in the accessory medulla may have a role in transmitting light information or phase information to facilitate coupling of the circadian clock.
The Drosophila orcokinin A isoform was observed to express in five pairs of neurons in the abdominal neuromeres of the larval CNS (Fig. 4). In Bombyx, orcokinin is widely distributed in the CNS and a pair of orcokinin-expressing neurosecretory cells in the prothoracic ganglion project their axons to the prothoracic gland to exert prothoracicotropic activity [32]. Although we were not able to accurately trace the axons of orcokinin-expressing neurons in the Drosophila larva by reporter expression driven by the orcokinin-GAL4 transgene, we did not observe GFP-labeled neural
J. Chen et al. / Peptides 68 (2015) 50–57
processes that terminate at the Drosophila larval ring gland, which corresponds to the Bombyx prothoracic gland. Further genetic approaches are necessary to elucidate the physiological functions of orcokinin in the nervous system. The Drosophila orcokinin B isoform is expressed in many endocrine cells in the anterior and middle midgut. A recent study showed that Drosophila orcokinin B is expressed in enteroendocrine cells that express allatostatin C [31]. As is the case for the many neuropeptides expressed in the intestine, further research is necessary to gain insight into the functions of orcokinin in the intestine. We observed that orcokinin B is expressed in a single unpaired cell in the abdominal ganglion of the adult CNS. In over 50 samples, the orcokinin B neuron in the abdominal ganglion was always unpaired. This is a unique expression pattern that may be linked to the expression and function of orcokinin in the midgut, since some neurons from the abdominal ganglion have been shown to innervate the intestine [4]. In summary, we provide comprehensive evidence through in situ hybridization, immunostaining, and an orcokinin-GAL4 driver that the two forms of Drosophila orcokinins are mainly expressed in different organs. Our findings provide a starting point for further molecular and genetic studies on the functions of each orcokinin, as well as the functional relationships between orcokinins and other regulatory peptides. Acknowledgements We would like to thank Barry J. Dickson (Janelia Farm Research Campus) for reagents. The Developmental Studies Hybridoma Bank (University of Iowa) provided the anti-Prospero antibody. This research was supported by Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea (NRF-2010-0010597 (J.Y.K), NRF-2013R1A1A2061120 (J.Y.K), NRF-2013R1A1A2010475 (Y.-J.K.)). References [1] Chen Y, Veenstra JA, Davis NT, Hagedorn HH. A comparative study of leucokininimmunoreactive neurons in insects. Cell Tissue Res 1994;276:69–83. [2] Christie AE. In silico analyses of peptide paracrines/hormones in Aphidoidea. Gen Comp Endocrinol 2008;159:67–79. [3] Clynen E, Schoofs L. Peptidomic survey of the locust neuroendocrine system. Insect Biochem Mol Biol 2009;39:491–507. [4] Cognigni P, Bailey AP, Miguel-Aliaga I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab 2011;13:92–104. [5] Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 2004;166:1775–82. [6] Hewes RS, Taghert PH. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 2001;11:1126–42. [7] Hofer S, Dircksen H, Tollback P, Homberg U. Novel insect orcokinins: characterization and neuronal distribution in the brains of selected dicondylian insects. J Comp Neurol 2005;490:57–71.
57
[8] Hofer S, Homberg U. Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae. J Exp Biol 2006;209:2794–803. [9] Hofer S, Homberg U. Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae. Cell Tissue Res 2006;325:589–600. [10] Hummon AB, Amare A, Sweedler JV. Discovering new invertebrate neuropeptides using mass spectrometry. Mass Spectrom Rev 2006;25:77–98. [11] Huybrechts J, Bonhomme J, Minoli S, Prunier-Leterme N, Dombrovsky A, AbdelLatief M, et al. Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum. Insect Mol Biol 2010;19(Suppl. 2):87–95. [12] Ito K, Shinomiya K, Ito M, Armstrong JD, Boyan G, Hartenstein V, et al. A systematic nomenclature for the insect brain. Neuron 2014;81: 755–65. [13] Jung SH, Lee JH, Chae HS, Seong JY, Park Y, Park ZY, et al. Identification of a novel insect neuropeptide, CNMa and its receptor. FEBS Lett 2014;588: 2037–41. [14] Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, Clark JM, et al. Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle. Proc Natl Acad Sci U S A 2010;107:12168–73. [15] Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 1999;22:451–61. [16] Liu F, Baggerman G, D’Hertog W, Verleyen P, Schoofs L, Wets G. In silico identification of new secretory peptide genes in Drosophila melanogaster. Mol Cell Proteomics 2006;5:510–22. [17] Nässel DR, Winther AM. Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 2010;92:42–104. [18] Ons S, Richter F, Urlaub H, Pomar RR. The neuropeptidome of Rhodnius prolixus brain. Proteomics 2009;9:788–92. [19] Park D, Veenstra JA, Park JH, Taghert PH. Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS ONE 2008;3:e1896. [20] Park JH, Kwon JY. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells. PLoS ONE 2011;6:e29022. [21] Park JH, Kwon JY. A systematic analysis of Drosophila gustatory receptor gene expression in abdominal neurons which project to the central nervous system. Mol Cells 2011;32:375–81. [22] Pascual N, Castresana J, Valero ML, Andreu D, Bellés X. Orcokinins in insects and other invertebrates. Insect Biochem Mol Biol 2004;34:1141–6. [23] Python F, Stocker R. Adult-like complexity of the larval antennal lobe of D. melanogaster despite markedly low numbers of odorant receptor neurons. J Comp Neurol 2002;445:374–87. [24] Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 1999;99:791–802. [25] Roller L, Yamanaka N, Watanabe K, Daubnerova I, Zitnan D, Kataoka H, et al. The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem Mol Biol 2008;38:1147–57. [26] Sharma Y, Cheung U, Larsen EW, Eberl DF. pPTGAL, a convenient Gal4 P-element vector for testing expression of enhancer fragments in Drosophila. Genesis 2002;34:115–8. [27] Soehler S, Stengl M, Reischig T. Circadian pacemaker coupling by multipeptidergic neurons in the cockroach Leucophaea maderae. Cell Tissue Res 2011;343:559–77. [28] Stangier J, Hilbich C, Burdzik S, Keller R. Orcokinin: a novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus. Peptides 1992;13:859–64. [29] Sterkel M, Oliveira PL, Urlaub H, Hernandez-Martinez S, Rivera-Pomar R, Ons S. OKB, a novel family of brain-gut neuropeptides from insects. Insect Biochem Mol Biol 2012;42:466–73. [30] Strand FL. Neuropeptides: regulators of physiological processes. Cambridge, MA: MIT Press; 1999. [31] Veenstra JA, Ida T. More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2. Cell Tissue Res 2014;357:607–21. [32] Yamanaka N, Roller L, Zitnan D, Satake H, Mizoguchi A, Kataoka H, et al. Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis. J Comp Neurol 2011;519:238–46.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster Ji Chen a , Min Sung Choi a , Akira Mizoguchi b , Jan A. Veenstra c , KyeongJin Kang d , Young-Joon Kim e,∗ , Jae Young Kwon a,∗∗ a
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan c Université de Bordeaux, INCIA UMR 5287 CNRS, 33405 Talence, France d Samsung Biomedical Research Institute, Department of Anatomy and Cell Biology, School of Medicine, Sungkyunkwan University, Suwon 440-746, Republic of Korea e School of Life Sciences, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 4 January 2015 Accepted 7 January 2015 Available online 16 January 2015 Keywords: Orcokinin Neuropeptide Drosophila melanogaster
a b s t r a c t Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1–5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model. © 2015 Elsevier Inc. All rights reserved.
Introduction Neuropeptides are an evolutionarily ancient class of signaling molecules that function as neurohormones, neuromodulators, and neurotransmitters, and regulate various developmental and physiological processes in almost all animal species [17,30]. The fruit fly Drosophila melanogaster genome has at least 45 neuropeptide genes, which encode much larger numbers of mature
∗ Corresponding author at: School of Life Sciences, GIST (Gwangju Institute of Science and Technology), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea. Tel.: +82 62 970 2492. ∗∗ Corresponding author at: Department of Biological Sciences, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea. Tel.: +82 31 299 4495. E-mail addresses: [email protected] (Y.-J. Kim), [email protected] (J.Y. Kwon). http://dx.doi.org/10.1016/j.peptides.2015.01.002 0196-9781/© 2015 Elsevier Inc. All rights reserved.
neuropeptides [6,13,17]. The number of genes encoding neuropeptides continues to increase as additional genes and mRNA species from animal genomes are uncovered thanks to advances in bioinformatical tools and RNA sequencing technology. Like other eukaryotic genes, a neuropeptide gene can produce multiple splicing variants, and alternative mRNA splicing further increases the repertoire of mature neuropeptides. Orcokinins were first identified as a myostimulatory factor in the hindgut of the crayfish Orconectes limosus [28]. Classical biochemical isolation, mass spectrometry, and most recently bioinformatic approaches have subsequently identified orcokinin-like peptides in numerous insect species [2,3,7,10,11,14,16,18,22,25]. The physiological function of orcokinins has been studied in several insect species. Orcokinins appear to have a role in the circadian clock controlling locomotory activity in the cockroach Leucophaea maderae [8], and act as neuronal prothoracicotropic factors in Bombyx mori [32]. A recent study revealed that the orcokinin
J. Chen et al. / Peptides 68 (2015) 50–57
gene produces at least two alternatively spliced mRNA transcripts that encode specific peptides in major orders of insect species, including Diptera (Anopheles gambiae), Lepitoptera (Bombyx mori), Hymenoptera (Apis mellifera), and Hemiptera (Rhodnius prolixus) [22,29,32]. According to these analyses [29,32], orcokinin A appears to occur mainly in the central nervous system (CNS), and orcokinin B in the CNS and anterior midgut. Two alternatively spliced forms of orcokinin were also shown to exist in D. melanogaster [31]. This study revealed that an antiserum against the predicted mature orcokinin B peptide showed immunoreactivity in the midgut of both larvae and adults. However, the neuronal expression patterns of the two orcokinin isoforms in Drosophila remain unknown. Here, we characterize the expression pattern of the two orcokinin isoforms in Drosophila using isoform-specific mRNA and protein staining, and transgenic orcokinin-GAL4 flies. The differential expression patterns of the two alternative mRNA splicing variants of the orcokinin gene suggest that the two isoforms have likely evolved to mediate different physiological functions. Materials and methods Drosophila stocks Flies were grown on standard cornmeal/agar culture medium at an average culture temperature of 23 ◦ C. To construct orcokininGAL4 transgenic flies, the orcokinin-GAL4 transgene was first prepared by cloning the 5 upstream region of orcokinin (−504 to +26; +1 indicates the transcription start site) amplified with the forward primer (5 -tatagcggccgcgcttgcagatgaatgggatt-3 ) and reverse primer (5 -gcgcgctctagaaccgccaggagcacatatag-3 ) into the pAGAL4 vector. This transgene was then inserted into a specific site on the second chromosome (VIE-72A, a gift from B.J. Dickson) using the C31 system [5]. pAGAL4 was prepared by inserting a sitespecific integration site (attB) into the 7–74 site of the pPTGAL4(+) vector [26]. For in situ hybridization and immunostaining, w1118 and orcokinin-GAL4; UAS-mCD8-GFP flies were used. UAS-mCD8GFP [15] and pdf-GAL4 [24] were described previously. Due to no obvious gender-specific differences in orcokinin expression (data not shown), we present data obtained from males. In situ hybridization The CNS and intestinal tissues from 3rd instar larvae or 5 to 9 day-old adults were dissected in phosphate-buffered saline with 0.2% Triton X-100 (PBS-T). For the adult intestines, whole abdomens with incisions were fixed and subjected to subsequent staining steps, and the intestine was dissected out for mounting. Samples were fixed in 4% paraformaldehyde in PBS-T for 2–4 h at room temperature, and washed with PBS-T. Proteinase K (50 g/ml in PBS-T) treatment was performed for 10 min, and then stopped by adding glycine-PBS-T (2 mg glycine/ml PBST). After washing with PBS-T, the tissues were postfixed in 4% paraformaldehyde in PBS-T for 1 h and washed with PBS-T. After prehybridization in hybridization solution (50% formamide, 50 g/ml heparin, 100 g/ml salmon testes DNA, 5× SSC, 0.1% Tween 20) for 10–20 min at 48 ◦ C, the dissected tissues were incubated with the ssDNA probe in hybridization solution for at least 20 h at 48 ◦ C. Digoxygenin (DIG)-labeled ssDNA probes targeting exons specific to each orcokinin isoform were prepared using the PCR-DIG Probe Synthesis kit (Roche). The following PCR primers were used to generate probes targeting the isoform-specific exons: orcokinin A-specific exon (+116 to +384; forward primer 5 -attgatttcggctgccaatggac-3 , reverse primer 5 ctagacgagctggttgaggatgc-3 ), and orcokinin B-specific exon (+153 to +402; forward primer 5 -atctgccagcgttaagagaattg-3 , reverse
51
primer, 5 -ctagtcgcttaggaactgtctac-3 ). The probe was mixed into hybridization solution (10 l of probe in 90 l of hybridization solution) and boiled for 40–45 min just before hybridization. After hybridization, the dissected tissues were washed with hybridization solution for 2–4 h at 48 ◦ C, and briefly washed with 1:1 hybridization solution: PBS-T and PBS-T only. The samples were then blocked with 3% goat serum in PBS-T for 30 min at room temperature. Two methods were used for amplification of the digoxygenin signal. The first method used alkaline phosphatase-labeled sheep anti-digoxygenin antibody (1:1000, Roche, Mannheim, Germany) to amplify the digoxygenin signal overnight at 4 ◦ C. Tissues were subsequently stained with nitro blue tetrazolium and 5-bromo4-chloro-3-indolyl-phosphate solution (NBT-BCIP, Roche) diluted 1:50 in alkaline phosphatase buffer (100 mM Tris, 50 mM MgCl2 , 100 mM NaCl, 0.1% Tween 20, pH 9.5). The second method utilized fluorophore-conjugated tyramide to amplify the digoxygenin signal. A tyramide signal amplification kit (Molecular Probes, Cat. No. T20924) was used following the manufacturer’s instructions. Briefly, rabbit monoclonal digoxigenin antibody (1:1000, Invitrogen, Cat. No. 700772) was added and the samples incubated overnight at 4 ◦ C. Samples were washed three times with PBS-T and 3% goat serum in PBS-T 30 min, before adding the goat antirabbit HRP-conjugated secondary antibody (1:200) for incubation at room temperature for at least 1 h. After three washes with PBS-T, Alexa Fluor® 568-labeled tyramide was added for 30 min at room temperature. Samples were then washed and mounted on a slide. The tyramide signal amplification method has the advantage of enabling double labeling of the sample with another antibody and simultaneous detection of fluorescent signals. When double labeling with orcokinin antibodies was performed, primary antibodies were added after the steps described above, and subsequent steps followed the immunostaining protocol described below. Immunostaining For the immunohistochemistry of intestinal cells, whole abdomens were stained and the stained intestines were dissected out for mounting before imaging. A previous immunocytochemistry protocol was followed [20], with minor modifications. Briefly, whole abdomens were dissected in phosphate buffered saline with 0.2% Triton X-100 (PBS-T, pH7.2), and fixed in 4% paraformaldehyde in PBS-T buffer for 2 h on ice. After 3 washes with PBS-T, samples were blocked with 3% goat serum in PBS-T for at least 30 min. Abdomens were incubated with primary antibodies or antiserum for 1–2 days at 4 ◦ C, washed with PBS-T, and incubated with secondary antibodies overnight at 4 ◦ C. For the immunohistochemistry of the brain and ventral nerve cord (VNC), adult brains and VNC were dissected and prepared as described [21,23]. Samples were incubated with primary antibodies or antiserum for 48 h at 4 ◦ C, washed with PBS-T, and incubated with secondary antibodies overnight at 4 ◦ C. The primary antibodies or antiserum used were rabbit antiGFP (1:1000, Molecular Probes), mouse anti-Bombyx orcokinin A (1:1000 [32]), mouse anti-Drosophila orcokinin B (1:1000 [31]), rabbit anti-leucokinin (1:200 [1]), and mouse anti-Prospero (1:10, Developmental Studies Hybridoma Bank at the University of Iowa). Prospero is a marker for enteroendocrine cells. The secondary antibodies used were goat anti-mouse and goat anti-rabbit IgG conjugated to either Alexa Fluor® 568 or Alexa Fluor® 488 (1:1000, Molecular Probes). Imaging and microscopy A Zeiss LSM 510, 700 laser-scanning confocal microscope was used to image fluorescent samples, and a Leica DM2500 microscope
52
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 1. Drosophila orcokinin gene structure. The Drosophila genome database (http://www.flybase.org) predicts different mRNA variants (orcokinin-RA and -RC), each of which encodes either of two mature orcokinin isoforms. Gray and red colored boxes indicate the untranslated and translated regions of orcokinin transcripts, respectively. The genomic fragment used to generate orcokinin-GAL4 is indicated by a black box. The regions that correspond to fully processed mature orcokinin isoforms are marked as thick black lines below the exon. The single-letter amino acid sequence of each mature isoform is shown in parentheses. Isoform-specific in situ probes were prepared from isoform-specific exons indicated by a blue (isoform A) or green box (isoform B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
with a digital camera (Canon EOS 700D) was used to image in situ hybridized samples. Results Two isoforms of orcokinin are predicted in the Drosophila genome According to the most recent version of the Drosophila genome database (FlyBase Release 6), the Drosophila orcokinin gene is predicted to produce mRNA splicing variants, each of which produces either of two mature orcokinin isoforms with distinctly different amino acid sequences (Fig. 1). Since studies in other insects suggested that orcokinin A and orcokinin B have distinct expression patterns in the CNS and intestine [29], we characterized the expression of both Drosophila isoforms in those tissues. For this purpose, we examined the expression pattern of an orcokinin-GAL4 driver, as well as endogenous expression of orcokinin A and orcokinin B using in situ hybridization and isoform-specific antisera, in the CNS and intestine. Transcripts specific for Drosophila orcokinin A and B share a common 5 upstream region, and the entire 5 region upstream of the translation initiation codon of the orcokinin gene was combined with the GAL4 open reading frame to construct the orcokinin-GAL4 driver (black box in Fig. 1). Exons specific to each isoform were amplified to prepare probes for in situ hybridization (green and blue boxes in Fig. 1). Orcokinin A is expressed in the CNS, but not in the intestine A digoxygenin-labeled ssDNA probe targeting the orcokinin A-specific exon was used to examine orcokinin A mRNA expression in the adult CNS. In situ hybridization in the adult fly brain labeled one pair of cell bodies near the center of the brain and four additional pairs near the optic lobes (Fig. 2A). The cell bodies located near the center of the brain were much larger in size, and appear to be located in the posterior region of ventrolateral neuropils, based on comparison of DIC images with 3D stacks of the adult brain provided by Virtual Fly Brain (http://www.virtualflybrain.org/site/stacks/index.htm [12]) (Fig. 2A). Thus, we named the larger cells orcoA-PLP (orcokinin Apositive posteriorlateral protocerebrum neurons). The four pairs of cells occurring laterally in the brain near the accessory medulla are closely positioned to the LNv cells which express pdf-GAL4, but are not the LNv cells per se (Fig. 2F and G). Thus, we named the four small neurons orcoA-AME (orcokinin A-positive accessory medulla neurons). The same in situ labeling was observed in
the adult brain regardless of whether alkaline phosphatase or a fluorophore-conjugated tyramide was used to amplify the digoxygenin signal (Fig. 2B). Drosophila orcokinin A expression examined by an antiserum against Bombyx orcokinin A showed an expression pattern identical to in situ mRNA staining (Fig. 2C). GFP expression driven by the orcokinin-GAL4 transgene was broader than the expression observed by in situ hybridization or antibody staining (Fig. 2D), likely reflecting some ectopic expression due to limitations of the GAL4-UAS system. Nevertheless, all adult brain cells positive for anti-orcokinin A staining overlapped with orcokininGAL4 transgene-expressing cells (Fig. 2D and E). Two pairs of cell bodies are clearly labeled in the adult ventral nerve cord (VNC) by both in situ hybridization and anti-orcokinin A staining (Fig. 3A and B), and all of these cells overlap with orcokininGAL4 transgene-expressing cells (Fig. 3C). Ectopic expression of the orcokinin-GAL4 transgene is also observed in the VNC (Fig. 3C). The orcokinin A-expressing cell bodies are located dorsally between the metathoracic neuromeres and abdominal ganglion, and were named orcoA-VNC. In the larval central nervous system (CNS), five pairs of abdominal neurons (orcokinin A-positive lateral abdominal neurons, or orcoA-LA) were specifically labeled by in situ hybridization specific for orcokinin A mRNA (Fig. 4A), anti-orcokinin A antibody staining (Fig. 4B), and orcokinin-GAL4 transgene expression (Fig. 4C). Each pair of orcoA-LA occurs laterally in each of the 5 abdominal neuromeres, reminiscent of the expression pattern of another neuropeptide drosokinin (aka Drosophila leucokinin), which also occurs in laterally paired neurons in the abdominal neuromeres A1–A7 [19]. Thus, we compared orcokinin A and drosokinin-expressing cells by double antibody staining, and found that the orcoA-LA neurons in the abdominal neuromeres are juxtaposed to those expressing drosokinin (Fig. 4D). In contrast to the expression in the CNS, larval and adult intestines are completely devoid of orcokinin A expression when examined with in situ hybridization and antibody staining (data not shown). Orcokinin B occurs mainly in the midgut enteroendocrine cells In situ hybridization of an orcokinin B-specific exon failed to detect orcokinin B mRNA-positive neurons in the adult CNS, except for one unpaired large cell in the abdominal ganglion (orcokinin Bpositive unpaired AG neuron or orcoB-uAG) (Fig. 5A). Likewise, the orcoB-uAG cell is the only cell in the CNS labeled by anti-orcokinin B staining (Fig. 5B). Cells expressing the orcokinin-GAL4 transgene also included orcoB-uAG, despite some ectopic expression in the abdominal ganglion (Fig. 5C). Unlike the A-isoform, the B-isoform
J. Chen et al. / Peptides 68 (2015) 50–57
53
Fig. 2. Drosophila orcokinin A expression in the adult brain. (A) In situ hybridization to orcokinin A shows expression in one pair of cell bodies near the center of the brain (orcoA-PLP) and 4 pairs of cell bodies in the accessory medulla (orcoA-AME). A, A , A are all images of the same adult fly brain. A is at an optic plane more posterior than A . A is a magnified image of the white box in A . The arrowheads in A indicate the cell bodies of four neurons expressing orcokinin A in the accessory medulla. (B) Fluorescence in situ hybridization, (C) orcokinin A antibody staining, and (D) orcokinin-GAL4 driven GFP reporter expression confirm the adult brain expression pattern observed in A. (D) All cells detected in the adult brain by orcokinin A antibody staining overlap with orcokinin-GAL4 transgene-expressing cells. (E) shows magnified images of the white boxes in D. (F) The 4 pairs of cells expressing orcokinin A in the accessory medulla are different cells from the LNv cells which express the pdf-GAL4 reporter. (G) is a magnified image of the white box in F. All images are from 5 to 9 day-old adult males. Dorsal is to the top. The scale bar indicates 50 m in A, A , B, C, D and F, and 100 m in A , E and G.
was not detected in the larval CNS by in situ hybridization (data not shown). In contrast to orcokinin A, we observed that orcokinin B is expressed in both adult and larval intestines by in situ hybridization (Fig. 6A, data not shown). This is consistent with a previous report that showed the presence of orcokinin B immunoreactivity in intestinal endocrine cells [31]. In situ hybridization also revealed that orcokinin B is mainly expressed in the adult anterior and
middle midgut, in cells whose morphology and position are characteristic of enteroendocrine cells (Fig. 6A). Intestinal cells detected by orcokinin B antisera are also labeled by the orcokinin-GAL4 transgene, although some intestinal cells positive for orcokinin-GAL4 lack anti-orcokinin B immunoreactivity (Fig. 6B). Colocalization of Prospero, an enteroendocrine marker, and orcokinin-GAL4 provides further verification that orcokinin B is expressed in the enteroendocrine cells (Fig. 6C).
54
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 3. Drosophila orcokinin A expression in the adult ventral nerve cord. (A) Two pairs of cell bodies are labeled in the adult ventral nerve cord by in situ hybridization. (B) Orcokinin A antibody staining and fluorescence in situ hybridization label the same cells in the adult ventral nerve cord. (C) Orcokinin A antibody staining and orcokinin-GAL4 driven GFP reporter expression label the same cells (orcoA-VNC, arrowheads) in the adult ventral nerve cord. Anterior is to the top. All images are from 5 to 9 day-old adult males. All scale bars indicate 50 m.
Fig. 4. Drosophila orcokinin A expression in the larva CNS. (A) In situ hybridization to orcokinin A labeled 5 pairs of neurons in the larval CNS. A is a magnified image of the white box in A. Double labeling of orcokinin A by (B) antibody staining and fluorescence in situ hybridization, and (C) antibody staining and orcokinin-GAL4 driven GFP reporter expression confirm the expression in five pairs of cells. (D) Orcokinin A-expressing cells are different cells from drosokinin-expressing cells. All images are from 3rd instar larvae. All scale bars indicate 50 m.
J. Chen et al. / Peptides 68 (2015) 50–57
55
Fig. 5. Drosophila orcokinin B expression in the adult CNS. (A) In situ hybridization of orcokinin B showed expression in a single unpaired cell in the abdominal ganglion. A is a magnified image of the white box in A. (B) Double labeling of orcokinin B by antibody staining and fluorescence in situ hybridization confirm the expression in one cell in the abdominal ganglion. B is a magnified image of the white box in B. (C) Double labeling of orcokinin B by antibody staining and orcokinin-GAL4 driven GFP reporter expression confirm the expression in one unpaired cell in the abdominal ganglion. Arrowheads indicate the single cell co-expressing orcokinin B antibody staining and orcokinin-GAL4 driven GFP reporter expression (orcoB-uAG). Anterior is to the top. All images are from 5 to 9 day-old adult males. All scale bars indicate 50 m.
Discussion Here, we examine the mRNA and peptide expression of the two orcokinin isoforms in the Drosophila CNS and intestine by in situ hybridization and immunolabeling. Our analysis demonstrates that the two isoforms display specific and distinct expression patterns in the CNS and midgut enteroendocrine cells: orcokinin A is expressed in the CNS, and orcokinin B is mainly expressed in the midgut. We also establish and confirm that the expression of an orcokininGAL4 driver recapitulates that of orcokinin peptides and mRNAs in the CNS and enteroendocrine cells, providing a useful genetic tool to manipulate the activities of orcokinin-producing cells in future functional analyses. In a study of Bombyx orcokinin expression, orcokinin mRNA was observed in the nervous system and midgut enteroendocrine cells, whereas orcokinin immunoreactivity was detected only in the nervous system [32]. Since the in situ probe used in the Bombyx study was specific to exons common to both the orcokinin A and B isoforms, the in situ results likely reflect a composite of both A and B isoform RNA expression. Considering that the Bombyx orcokinin antibody was raised against an orcokinin A-specific region, the previous Bombyx study is consistent with the conclusions we made in this study: the A- and B-isoforms of orcokinin mainly occur in
the CNS and in the midgut, respectively. In Rhodnius prolixus, the orcokinin A isoform is expressed in the CNS, and the B isoform in the CNS as well as the anterior midgut [29]. In Drosophila, orcokinin B was reported to be expressed in the midgut of adults and larvae [31]. Together with this previous report, our results provide further support that the two alternative forms of orcokinin have evolved to function separately in two different tissues, the nervous system and the intestinal enteroendocrine system, across different insect species. Based on mRNA and peptide expression patterns, we can hypothesize that the Drosophila orcokinin A isoform mainly functions in the nervous system. Some clues to the role of orcokinin A in the accessory medulla of Drosophila can be inferred from studies in other insects. In the cockroach Leucophaea maderae, about 30 neurons in the accessory medulla are labeled by orcokinin immunostaining [9,27]. Four groups of neurons (MCI, MCII, MCIII, and MCIV) in the cockroach accessory medulla have been proposed to act in coupling the two bilaterally symmetric circadian pacemakers located in the bilateral accessory medullae [27]. PDFand orcokinin-reactive PDFMe neurons in the MCI group were proposed to transmit circadian phase information for coupling, and orcokinin-immunoreactive VMNe neurons in the MCII group were proposed to transmit light information to the circadian
56
J. Chen et al. / Peptides 68 (2015) 50–57
Fig. 6. Drosophila orcokinin B expression in the adult intestine. (A) In situ hybridization showed that orcokinin B is expressed in the adult anterior midgut, in cells whose morphology and position are characteristic of enteroendocrine cells. A and A are magnified images of the white box in A, and show images of different focal planes. The cells in A marked by arrowheads have a shape characteristic of enteroendocrine cells. (B) Double labeling of orcokinin B antibody and orcokinin-GAL4 driven GFP reporter expression confirm expression in the anterior midgut. (C) Cells labeled by orcokinin-GAL4 driven GFP reporter expression overlap with cells expressing Prospero, an enteroendocrine cell marker. All images are from 5 to 9 day-old adult males. The scale bar indicates 100 m in A and C, and 50 m in A , A and B.
clock [8,27]. Orcokinin injections into the vicinity of the accessory medulla caused phase shifts in circadian locomotor activity similar to phase shifts caused by light pulses, leading to the suggestion that orcokinin-related peptides play an important role in light entrainment of the circadian clock [8]. It thus appears to be a viable hypothesis that the Drosophila orcokinin A form-immunoreactive neurons in the accessory medulla may have a role in transmitting light information or phase information to facilitate coupling of the circadian clock.
The Drosophila orcokinin A isoform was observed to express in five pairs of neurons in the abdominal neuromeres of the larval CNS (Fig. 4). In Bombyx, orcokinin is widely distributed in the CNS and a pair of orcokinin-expressing neurosecretory cells in the prothoracic ganglion project their axons to the prothoracic gland to exert prothoracicotropic activity [32]. Although we were not able to accurately trace the axons of orcokinin-expressing neurons in the Drosophila larva by reporter expression driven by the orcokinin-GAL4 transgene, we did not observe GFP-labeled neural
J. Chen et al. / Peptides 68 (2015) 50–57
processes that terminate at the Drosophila larval ring gland, which corresponds to the Bombyx prothoracic gland. Further genetic approaches are necessary to elucidate the physiological functions of orcokinin in the nervous system. The Drosophila orcokinin B isoform is expressed in many endocrine cells in the anterior and middle midgut. A recent study showed that Drosophila orcokinin B is expressed in enteroendocrine cells that express allatostatin C [31]. As is the case for the many neuropeptides expressed in the intestine, further research is necessary to gain insight into the functions of orcokinin in the intestine. We observed that orcokinin B is expressed in a single unpaired cell in the abdominal ganglion of the adult CNS. In over 50 samples, the orcokinin B neuron in the abdominal ganglion was always unpaired. This is a unique expression pattern that may be linked to the expression and function of orcokinin in the midgut, since some neurons from the abdominal ganglion have been shown to innervate the intestine [4]. In summary, we provide comprehensive evidence through in situ hybridization, immunostaining, and an orcokinin-GAL4 driver that the two forms of Drosophila orcokinins are mainly expressed in different organs. Our findings provide a starting point for further molecular and genetic studies on the functions of each orcokinin, as well as the functional relationships between orcokinins and other regulatory peptides. Acknowledgements We would like to thank Barry J. Dickson (Janelia Farm Research Campus) for reagents. The Developmental Studies Hybridoma Bank (University of Iowa) provided the anti-Prospero antibody. This research was supported by Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea (NRF-2010-0010597 (J.Y.K), NRF-2013R1A1A2061120 (J.Y.K), NRF-2013R1A1A2010475 (Y.-J.K.)). References [1] Chen Y, Veenstra JA, Davis NT, Hagedorn HH. A comparative study of leucokininimmunoreactive neurons in insects. Cell Tissue Res 1994;276:69–83. [2] Christie AE. In silico analyses of peptide paracrines/hormones in Aphidoidea. Gen Comp Endocrinol 2008;159:67–79. [3] Clynen E, Schoofs L. Peptidomic survey of the locust neuroendocrine system. Insect Biochem Mol Biol 2009;39:491–507. [4] Cognigni P, Bailey AP, Miguel-Aliaga I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab 2011;13:92–104. [5] Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 2004;166:1775–82. [6] Hewes RS, Taghert PH. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 2001;11:1126–42. [7] Hofer S, Dircksen H, Tollback P, Homberg U. Novel insect orcokinins: characterization and neuronal distribution in the brains of selected dicondylian insects. J Comp Neurol 2005;490:57–71.
57
[8] Hofer S, Homberg U. Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae. J Exp Biol 2006;209:2794–803. [9] Hofer S, Homberg U. Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae. Cell Tissue Res 2006;325:589–600. [10] Hummon AB, Amare A, Sweedler JV. Discovering new invertebrate neuropeptides using mass spectrometry. Mass Spectrom Rev 2006;25:77–98. [11] Huybrechts J, Bonhomme J, Minoli S, Prunier-Leterme N, Dombrovsky A, AbdelLatief M, et al. Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum. Insect Mol Biol 2010;19(Suppl. 2):87–95. [12] Ito K, Shinomiya K, Ito M, Armstrong JD, Boyan G, Hartenstein V, et al. A systematic nomenclature for the insect brain. Neuron 2014;81: 755–65. [13] Jung SH, Lee JH, Chae HS, Seong JY, Park Y, Park ZY, et al. Identification of a novel insect neuropeptide, CNMa and its receptor. FEBS Lett 2014;588: 2037–41. [14] Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, Clark JM, et al. Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle. Proc Natl Acad Sci U S A 2010;107:12168–73. [15] Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 1999;22:451–61. [16] Liu F, Baggerman G, D’Hertog W, Verleyen P, Schoofs L, Wets G. In silico identification of new secretory peptide genes in Drosophila melanogaster. Mol Cell Proteomics 2006;5:510–22. [17] Nässel DR, Winther AM. Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 2010;92:42–104. [18] Ons S, Richter F, Urlaub H, Pomar RR. The neuropeptidome of Rhodnius prolixus brain. Proteomics 2009;9:788–92. [19] Park D, Veenstra JA, Park JH, Taghert PH. Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS ONE 2008;3:e1896. [20] Park JH, Kwon JY. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells. PLoS ONE 2011;6:e29022. [21] Park JH, Kwon JY. A systematic analysis of Drosophila gustatory receptor gene expression in abdominal neurons which project to the central nervous system. Mol Cells 2011;32:375–81. [22] Pascual N, Castresana J, Valero ML, Andreu D, Bellés X. Orcokinins in insects and other invertebrates. Insect Biochem Mol Biol 2004;34:1141–6. [23] Python F, Stocker R. Adult-like complexity of the larval antennal lobe of D. melanogaster despite markedly low numbers of odorant receptor neurons. J Comp Neurol 2002;445:374–87. [24] Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 1999;99:791–802. [25] Roller L, Yamanaka N, Watanabe K, Daubnerova I, Zitnan D, Kataoka H, et al. The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem Mol Biol 2008;38:1147–57. [26] Sharma Y, Cheung U, Larsen EW, Eberl DF. pPTGAL, a convenient Gal4 P-element vector for testing expression of enhancer fragments in Drosophila. Genesis 2002;34:115–8. [27] Soehler S, Stengl M, Reischig T. Circadian pacemaker coupling by multipeptidergic neurons in the cockroach Leucophaea maderae. Cell Tissue Res 2011;343:559–77. [28] Stangier J, Hilbich C, Burdzik S, Keller R. Orcokinin: a novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus. Peptides 1992;13:859–64. [29] Sterkel M, Oliveira PL, Urlaub H, Hernandez-Martinez S, Rivera-Pomar R, Ons S. OKB, a novel family of brain-gut neuropeptides from insects. Insect Biochem Mol Biol 2012;42:466–73. [30] Strand FL. Neuropeptides: regulators of physiological processes. Cambridge, MA: MIT Press; 1999. [31] Veenstra JA, Ida T. More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2. Cell Tissue Res 2014;357:607–21. [32] Yamanaka N, Roller L, Zitnan D, Satake H, Mizoguchi A, Kataoka H, et al. Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis. J Comp Neurol 2011;519:238–46.