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Antisera to multiple antigenic peptides detect neuropeptide processing
Neuropeptides (1999) 33 (1), 35–40 © 1999 Harcourt Brace & Co. Ltd
Antisera to multiple antigenic peptides detect neuropeptide processing R. Nichols,1 I. Lim,2 J. McCormick3 Department of Biological Chemistry, University of Michigan, Ann Arbor, USA Undergraduate Honors Program, University of Michigan, Ann Arbor, USA 3Department of Biology, University of Michigan, Ann Arbor, USA 1 2
Summary Peptides act as critical messengers of essential physiological function. Frequently, several peptides are encoded in the same precursor and, often, there is structure relatedness among the gene products. The complexity of protein precursors and presence of homologous peptides raises issues about regulation of gene expression and function of structurally-related peptides. We have determined the cellular location of DPKQDFMRFamide and SDNFMRFamide encoded in the Drosophila FMRFamide gene. We raised antisera that distinguish between the two peptides and conducted double label immunostaining utilizing antisera raised in the same host species. We found that DPKQDFMRFamide and SDNFMRFamide are present in distinct distribution patterns. We also established that the peptides are present in cells stained by FMRFamide antisera. Thus, our data are consistent with the conclusion that Drosophila contains cell-specific proteolytic processing enzymes capable of posttranslationally cleaving a polypeptide protein precursor to yield unique expression patterns of neuropeptides that may have diverse activities.
INTRODUCTION Peptides that serve as critical physiological messengers are frequently contained in a protein precursor with other structurally-related gene products. In order to decipher peptide signaling pathways, it is important to elucidate regulation of gene expression, in particular, for polypeptide protein precursors. One mode of regulation is differential posttranslational processing of precursors by cell-specific proteolytic enzymes to produce diverse expression patterns for structurally similar gene products. Unique distributions can suggest that the homologous peptides have dissimilar activities. Therefore, determining the expression patterns of peptides present in a complex protein precursor provides important information related to gene regulation and function. Peptides with a common C-terminal FMRFamide but distinct N-terminal amino acid extensions are present throughout the animal kingdom.1,2 Since the discovery of Received 24 August 1998 Accepted 23 January 1999 Correspondence to: R. Nichols, Department of Biological Chemistry, 830 N. University St., University of Michigan, Ann Arbor, MI 48109–1048, USA. Tel: 734 764 4467; Fax: 734 647 0884; E-mail: [email protected]
FMRFamide as a cardioexcitatory molecule,3 a vast number of structurally-related peptides has been identified. Typically, organisms have multiple genes containing numerous FMRFamide peptides.4–7 In Drosophila melanogaster, drosulfakinin, Dsk,8,9 dromyosuppressin, Dms,10 and FMRFamide,11,12 encode several peptides with structural homology to FMRFamide. We have reported the isolation of the structurally-related peptides DPKQDFMRFamide and SDNFMRFamide encoded in the FMRFamide gene.10 Although FMRFamide-related peptides constitute a major class of messengers, relatively little is known about regulation of gene expression and signaling of this neuropeptide family. Data indicate that FMRFamide peptides may act directly through peptide-gated ion channels13–15 as well as G protein-coupled receptors,16 thus, multiple transduction pathways exist for these neuropeptides. Determining the cells in which these peptides are present is important in deciphering FMRFamide neurotransmission. Here, we report the distribution of Drosophila DPKQDFMRFamide and SDNFMRFamide in neural tissue. In order to investigate FMRFamide gene regulation, we raised antisera to distinguish between 35
36 Nichols et al.
DPKQDFMRFamide and SDNFMRFamide. We designed each antigen as a multiple antigenic peptide (MAP)17,18 to the N-terminal extensions of the two structurally-related peptides. Utilizing a double immunolabeling protocol for antisera generated in the same host species, we mapped the neural distribution of the neuropeptides. We observed that DPKQDFMRFamide and SDNFMRFamide have distinct, non-overlapping cellular expression patterns suggesting that the precursor is differentially processed. Thus, our data support the conclusion that Drosophila contains cell-specific proteolytic enzymes to posttranslationally cleave a polypeptide protein precursor producing structurally-related neuropeptides that may be functionally dissimilar.
MATERIALS AND METHODS Synthesis and characterization of antigens The antigens, DPKQD-MAP and SDNFM-MAP, were each synthesized as a multiple antigenic peptide (MAP), a core matrix of branching lysine onto which the peptide was attached through the carboxyl terminus at eight branch sites.17,18 Antisera production Antisera were raised in New Zealand white rabbits in accordance with institutional guidelines. Initial immunizations were subcutaneous injections at multiple sites of a total of 1 m/l of antigen emulsified in Freund’s complete adjuvant. Subsequent boosts were given every week by subcutaneous injections of 0.1–0.5 m/l of antigen in Freund’s incomplete adjuvant. Immunocytochemical grade FMRFamide antisera raised in rabbits were purchased from Peninsula Labs. Characterization of antisera Antisera were purified on peptide affinity columns made by coupling the appropriate antigen to Affi-gel 10 resin (Bio-Rad Labs) as previously described.19–21 Incubating antisera raised to DPKQD-MAP with DPKQDFMRFamide or DPKQD-MAP abolished all staining, while incubation with FMRFamide did not. Incubating antisera raised to SDNFM-MAP with SDNFM-MAP abolished all staining, while incubating with FMRFamide did not. Immunofluorescence protocol Indirect immunofluorescence was done as previously described.19 Whole mount tissue preparations of central nervous system tissue were prepared from third instar larvae. Tissue was dissected in cold, calcium-free Neuropeptides (1999) 33(1), 35–40
Drosophila Ringer’s solution (130 mM NaCl, 4.7 mM KCl, 1.8 mM MgCl2, 0.74 mM Na2H2PO4, pH 7), fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 4 h, rinsed for 1 h with 2–3 changes of PTN (0.1 M sodium phosphate, pH 7.4, 0.3% Triton X-100, 0.2% NaN3 with 0.1% BSA), and incubated in primary antisera overnight at 4°C. Tissue was rinsed for 1 h with 3–4 changes of PTN, incubated for 4–6 h in goat anti-rabbit secondary antibody, and rinsed for 3–4 h with 8–12 changes of PTN. Tissue was then rinsed for 30 min in 4 mM sodium carbonate, pH 9.5, and mounted in 80% glycerol in 100 mM sodium carbonate, pH 9.5, containing 5% n-propyl gallate for microscopy. Double-label immunofluorescence was done as described previously with the following modifications. After incubation with the first primary antisera and goat anti-rabbit Cy3-labeled Fab fragment (Jackson ImmunoResearch Labs), the tissue was rinsed in PTN for 2 h, and incubated in FITC-conjugated goat anti-rabbit secondary antibody (Sigma Chemical Co.) for 4–6 h. Tissue was then washed extensively and prepared for microscopy as described above. Microscopy and data collection Fluorescent signal was imaged with a Bio-Rad MRC600 Iaser scanning confocal microscope equipped with a Kr-Ar laser attached to a Nikon inverted microscope. Optical sections or z series were collected using the Comos program. Adobe photoshop software, version 3.0, was used to process the data and images were printed with a Kodak XLS8600 printer. The terminology used to identify the antisera stained cells is consistent with previous publications.19,22–24 Immunoreactivity was observed bilaterally symmetric to the midline such that reference to one cell indicates the presence of a pair of cells positioned bilaterally symmetric to one another. Signal intensity was strong and consistent, and eight or more preparations were analyzed for each experiment. Antisera raised to DPKQDMAP and SDNFM-MAP are referred to as DPKQDFMRFamide antisera and SDNFMRFamide antisera, respectively. RESULTS In the larval central nervous system tissue, DPKQDFMRFamide antisera stain two neurons in the superior protocerebrum (SP1 and SP2), two in the subesophageal ganglion (SE2 and Sv), one in each of the three thoracic ganglion (T1–3), one medial neuron in the second thoracic ganglion (T2dm), and one in an abdominal ganglion (A8). (Fig. 1) Immunoreactive processes from SP1 project to the medial commissure of the brain lobes. © 1999 Harcourt Brace & Co. Ltd
Antisera to multiple antigenic peptides 37
Fig. 1 DPKQDFMRFamide immunoreactive material in the larval central nervous system. Antisera to DPKQD-MAP stain cells in the superior protocerebrum (SP1 and SP2), the subesophageal ganglion (Sv and SE2), the thoracic ganglia (T1-3 and T2dm), and an abdominal ganglion (A8). The bar in the lower right-hand corner represents 50 microns.
Fig. 2 SDNFMRFamide immunoreactive material in the larval central nervous system. Antisera to SDNFM-MAP stain cells in the subesophageal ganglion (SE). The bar in the lower right-hand corner represents 50 microns.
Immunoreactive processes project from SE2 along the midline of the ventral ganglion. T1–3 each has an immunoreactive process that projects medially toward the midline. No immunoreactive process was observed from SP2, Sv, T2dm, or A8. Based on position and number of cells, DPKQDFMRFamide immunoreactive material is present in neurons that contain the FMRFamide precursor.24 SDNFMRFamide antisera stain several neurons in the subesophageal ganglion (SE) (Fig. 2). Immunoreactive processes originating from SE neurons project anteriorly along the midline to the superior protocerebrum where they extend laterally terminating in numerous varicosities. Processes extend from the SDNFMRFamide antisera stained neurons laterally to the subesophageal ganglion, then turn and project posteriorly to the ventral ganglion along the midline. Immunoreactive processes from SDNFMRFamide antisera stained neurons also extend laterally and anteriorly to the region near the esophagus.
Based on position and number of cells, SDNFMRFamide immunoreactive material is present in neurons that contain the FMRFamide precursor.24 DPKQDFMRFamide antisera stain a subset of neurons recognized by FMRFamide antisera (Fig. 3). DPKQDFMRFamide and FMRFamide immunoreactivity are present in SP1 and SP2 protocerebral neurons, SE2 and Sv subesophageal neurons, T1–3 and T2dm thoracic neurons, and the A8 abdominal neuron. SDNFMRFamide antisera also stain a subset of neurons recognized by FMRFamide antisera (Fig. 4). SDNFMRFamide and FMRFamide immunoreactivity are present in the SE subesophageal neurons. Double immunolabeling was done with DPKQDFMRFamide antisera and SDNFMRFamide antisera in the same tissue preparation (Fig. 5). Our results indicate that DPKQDFMRFamide and SDNFMRFamide expression patterns do not overlap. The two subesophageal neurons stained by DPKQDFMRFamide
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Fig. 3 DPKQDFMRFamide and FMRFamide immunoreactive materials in the larval central nervous system. A red fluorescent signal is the result of the Cy3-labeled secondary antibody used to recognize DPKQDFMRFamide antisera and a green fluorescent signal reflects the FITC-labeled second antibody used to recognize FMRFamide antisera. The yellow fluorescent signal indicates that both DPKQDFMRFamide and FMRFamide immunoreactive materials are present. The bar in the lower right-hand corner represent 50 microns.
Fig. 4 SDNFMRFamide and FMRFamide immunoreactive materials in the larval central nervous system. A red fluorescent signal is the result of the Cy3-labeled secondary antibody used to recognize SDNFMRFamide antisera and a green fluorescent signal reflects the FITC-labeled secondary antibody used to recognize FMRFamide antisera. The yellow fluorescent signal indicated that both SDNFMRFamide and FMRFamide immunoreactive materials are present. The bar in the lower right-hand corner represents 50 microns.
antisera are posterior to the cluster of neurons recognized by SDNFMRFamide antisera. The DPKQDFMRFamide-immunoreactive process from the subesophageal neuron that extends into the ventral ganglion along the midline is medial to the SDNFMRFamide-immunoreactive process (Fig. 5).
diverse biological activities. We are interested in regulation of expression of FMRFamide-related peptides. In this manuscript we report raising antisera to DPKQDFMRFamide and SDNFMRFamide and determining the distribution of the structurally-related neuropeptides. Double immunolabeling is critical for simultaneously visualizing two antigens in the same tissue preparation. Methods using antisera from the same species29–34 have general application, but can have drawbacks. Some protocols rely on preabsorption with normal serum to help avoid crossreactivity, requiring an additional procedural step with possible unsatisfactory results. Other procedures utilize enzymatic or chemical reactions that can result in high background. Linking a fluorescent or chemical tag to primary antisera requires manipulation of the antisera. We conducted a double labeling protocol using antisera raised to MAP antigens in different
DISCUSSION Regulation of neuropeptide signaling is critical to physiology, hence, several mechanisms exist for modulating neurotransmission. One mode of regulation is at the posttranslational level with cell-specific proteolytic processing enzymes cleaving a precursor to produce neuropeptides in unique expression patterns.25–28. Through differential processing structurally-related peptides present in the same precursor can have different cellular or developmental distribution patterns and Neuropeptides (1999) 33(1), 35–40
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Antisera to multiple antigenic peptides 39
structurally-related peptides encoded in the FMRFamide gene. Peptide-specific antisera that distinguish between structurally-related neuropeptides are crucial for establishing cellular distribution and identifying sites of action to decipher gene regulation and elucidate physiological function. In this study we determined that DPKQDFMRFamide antisera and SDNFMRFamide antisera stain a distinct pattern of cells and, thus, are not co-expressed. In addition, we show that DPKQDFMRFamide and SDNFMRFamide immunoreactivities are present in cells also stained by antisera to FMRFamide. The neurons stained by DPKQDFMRFamide antisera and SDNFMRFamide antisera are similar in distribution and number with those previously identified as containing proFMRFamide.24 Thus, these data suggest that the Drosophila FMRFamide gene undergoes differential processing to produce unique distributions of structurally similar peptides with different activities. ACKNOWLEDGEMENT Research grants from The National Science Foundation and The American Heart Association Michigan Affiliate to RN are acknowledged. REFERENCES Fig. 5 SDNFMRFamide and DPKQDFMRFamide immunoreactive materials in the larval central nervous system. Red fluorescent signal reflects the Cy3-labeled secondary antibody used to recognize SDNFMRFamide antisera and the green fluorescent signal reflects the FITC-labeled secondary antibody used to recognize DPKQDFMRFamide antisera. The absence of any yellow fluorescent signal indicates that DPKQDFMRFamide and SDNFMRFamide immunoreactive materials are not present in the same cells or processes. The bar in the lower right-hand corner represents 50 microns.
animals of the same host species. The first primary antisera applied are detected by fluorescently-labeled Fab fragment to prevent the binding of the second primary antisera to the first secondary antibody. The use of a MAP can be advantageous since it can be designed to a short length of sequence. Neuropeptides are frequently short in length and, if there is structure similarity, only a few amino acid residues may distinguish between peptides. Also, the use of a MAP does not require a carrier molecule which itself may be antigenic.17,18 Antisera to FMRFamide stain numerous cells in Drosophila neural tissue 11,12,22, however, because FMRFamide antisera recognize a common structure in numerous peptides, these data cannot be unambiguously interpreted. We raised antisera that distinguish between DPKQDFMRFamide and SDNFMRFamide, © 1999 Harcourt Brace & Co. Ltd
1. Raffa RB. The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals. Peptides 1988; 9: 915–922. 2. Price DA, Greenberg MJ. The hunting of the FaRPs: the distribution of FMRFamide-related peptides. Biol Bull 1989; 177: 198–205. 3. Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977; 197: 670–672. 4. Marks NJ, Shaw C, Maule AG et al. Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the presence of more than one FMRFamide-related peptideencoding gene. Biochem Biophys Res Commun 1995; 217: 845–851. 5. Price DA, Doble KE, Lesser W et al. The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current. Biol Bull 1996; 191: 341–352. 6. Edison AS, Messinger LA, Stretton AO. afp-1: a gene encoding multiple transcripts of a new class of FMRFamide-like neuropeptides in the nematode Ascaris suum Peptides 1997; 18: 929–935. 7. Nelson LS, Kim K, Memmott JE, Li C. FMRFamide-related gene family in the nematode, Caenorhabditis elegans. Mol Brain Res 1998; 58: 103–111. 8. Nichols R, Schneuwly SA, Dixon JE. Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. J Biol Chem 1988; 263: 12167–12170. 9. Nichols R. Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol Cell Neurosci 1992; 3: 342–347. 10. Nichols R. Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides. J Mol Neurosci 1992; 3: 213–218.
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11. Nambu JR, Murphy-Erdosh C, Andrews PC, Feistner GJ, Scheller RH. Isolation and characterization of a Drosophila neuropeptide gene. Neuron 1988; 1: 55–61. 12. Schneider LE, Taghert PH. Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide). Proc Natl Acad Sci 1988; 85: 1993–1997. 13. Lingueglia E, Champigny G, Lazdunski M, Barby P. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature 1995; 378: 730–733. 14. Cottrell G. The first peptide-gated ion channel. J exp Biol 1997; 200: 2377–2386. 15. Darboux I, Lingueglia E, Pauron D, Barbry P, Lazdunski M. A new member of the amiloride-sensitive sodium channel family in Drosophila melanogaster peripheral nervous system. Biochem Biophys Res Commun 1998; 246: 210–216. 16. Chin GJ, Payza KE, Lesser W, Greenberg MJ, Doble KE. Characterization and solubilization of the FMRFamide receptor of squid. Biol Bull 1994; 191: 341–352. 17. Posnett DN, Tam JP. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. In Langone JJ, ed. Methods in Enzymology. New York, Academic Press 1989; 178: 739–746. 18. Nichols R, McCormick J, Lim I. Multiple antigenic peptides designed to structurally-related Drosophila peptides. Peptides 1997; 18: 41–45. 19. McCormick J, Nichols R. Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster. J Comp Neurol 1990; 338: 279–288. 20. Nichols R, McCormick J, Lim I, Starkman J. Spatial and temporal analysis of the Drosophila FMRFamide neuropeptide gene product SDNFMRFamide: evidence for a restricted cellular expression pattern. Neuropeptides 1995; 29: 205–213. 21. Nichols R, McCormick J, Lim I, Caserta L. Cellular expression of the Drosophila neuropeptide, DPKQDFMRFamide: evidence for differential processing of the FMRFamide polypeptide precursor. J Mol Neurosci 1995; 6: 1–10. 22. White K, Hurteau P, Punsal P. Neuropeptide-FMRFamide-like immunoreactivity in Drosophila: Development and distribution. J Comp Neurol 1986; 247: 430–438. 23. Lundquist T, Nässel DR. Substance P-, FMRFamide- and gastrin/cholecystokinin-like immunoreactive neurons in the
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thoraco-abdominal ganglia of the flies Drosophila and Calliphora. J Comp Neurol 1990; 294: 161–178. Schneider LE, Sun ET, Garland DH, Taghert PH. An immunocytochemical study of the FMRFamide neuropeptide gene products in Drosophila. J Comp Neurol 1993; 337: 446–460. Curry WJ, Johnston CF, Hutton JC et al. The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera. Histochem 1991; 96: 531–538. LeBlanc AC, Chen HY, Autilio-Gambetti L, Gambetti P. Differential APP gene expression in rat cerebral cortex, meninges, and primary astroglial, microglial, and neuronal cultures. FEBS Lett. 1991; 292: 171–178. Brand SJ, Fuller PJ. Differential gastrin gene expression in rat gastrointestinal tract and pancreas during neonatal development. J Biol Chem 1993; 263: 5341–5347. Mineo I, Matsumura T, Shingu R, Namba M, Kuwajima M, Matsuzawa Y. The role of prohormone convertases PC1 (PC3) and PC2 in the cell-specific processing of proglucagon. Biochem Biophys Res Comm 1990; 207: 646–651. Valnes K, Brandtzaeg P. Comparison of paired immunofluorescence and paired immunoenzyme staining methods based on primary antisera from the same species. J Histochem Cytochem 1982; 30: 518–524. Van der Loos CM, Das PK, Van den Oord, JJ, Housthoff, HJ. Multiple immunoenzyme staining techniques. J Immunol Methods 1989; 117: 45–52. Tibbetts M, Nichols R. Immunocytochemistry of two sequencerelated neuropeptides in Drosophila. Neuropeptides 1993; 24: 321–325. Würden S, Homberg U. A simple method for immunofluorescent double staining with primary antisera from the same species. J Histochem Cytochem 1993; 41: 627–630. Negoescu A, Labat-Moleur F, Lorimier P et al. F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 1994; 42: 433–437. Eichmüller S, Stevenson PA, Paus R. A new method for double immunolabeling with primary antibodies from identical species. J Immunol Methods 1996; 190: 255–265.
© 1999 Harcourt Brace & Co. Ltd
Antisera to multiple antigenic peptides detect neuropeptide processing R. Nichols,1 I. Lim,2 J. McCormick3 Department of Biological Chemistry, University of Michigan, Ann Arbor, USA Undergraduate Honors Program, University of Michigan, Ann Arbor, USA 3Department of Biology, University of Michigan, Ann Arbor, USA 1 2
Summary Peptides act as critical messengers of essential physiological function. Frequently, several peptides are encoded in the same precursor and, often, there is structure relatedness among the gene products. The complexity of protein precursors and presence of homologous peptides raises issues about regulation of gene expression and function of structurally-related peptides. We have determined the cellular location of DPKQDFMRFamide and SDNFMRFamide encoded in the Drosophila FMRFamide gene. We raised antisera that distinguish between the two peptides and conducted double label immunostaining utilizing antisera raised in the same host species. We found that DPKQDFMRFamide and SDNFMRFamide are present in distinct distribution patterns. We also established that the peptides are present in cells stained by FMRFamide antisera. Thus, our data are consistent with the conclusion that Drosophila contains cell-specific proteolytic processing enzymes capable of posttranslationally cleaving a polypeptide protein precursor to yield unique expression patterns of neuropeptides that may have diverse activities.
INTRODUCTION Peptides that serve as critical physiological messengers are frequently contained in a protein precursor with other structurally-related gene products. In order to decipher peptide signaling pathways, it is important to elucidate regulation of gene expression, in particular, for polypeptide protein precursors. One mode of regulation is differential posttranslational processing of precursors by cell-specific proteolytic enzymes to produce diverse expression patterns for structurally similar gene products. Unique distributions can suggest that the homologous peptides have dissimilar activities. Therefore, determining the expression patterns of peptides present in a complex protein precursor provides important information related to gene regulation and function. Peptides with a common C-terminal FMRFamide but distinct N-terminal amino acid extensions are present throughout the animal kingdom.1,2 Since the discovery of Received 24 August 1998 Accepted 23 January 1999 Correspondence to: R. Nichols, Department of Biological Chemistry, 830 N. University St., University of Michigan, Ann Arbor, MI 48109–1048, USA. Tel: 734 764 4467; Fax: 734 647 0884; E-mail: [email protected]
FMRFamide as a cardioexcitatory molecule,3 a vast number of structurally-related peptides has been identified. Typically, organisms have multiple genes containing numerous FMRFamide peptides.4–7 In Drosophila melanogaster, drosulfakinin, Dsk,8,9 dromyosuppressin, Dms,10 and FMRFamide,11,12 encode several peptides with structural homology to FMRFamide. We have reported the isolation of the structurally-related peptides DPKQDFMRFamide and SDNFMRFamide encoded in the FMRFamide gene.10 Although FMRFamide-related peptides constitute a major class of messengers, relatively little is known about regulation of gene expression and signaling of this neuropeptide family. Data indicate that FMRFamide peptides may act directly through peptide-gated ion channels13–15 as well as G protein-coupled receptors,16 thus, multiple transduction pathways exist for these neuropeptides. Determining the cells in which these peptides are present is important in deciphering FMRFamide neurotransmission. Here, we report the distribution of Drosophila DPKQDFMRFamide and SDNFMRFamide in neural tissue. In order to investigate FMRFamide gene regulation, we raised antisera to distinguish between 35
36 Nichols et al.
DPKQDFMRFamide and SDNFMRFamide. We designed each antigen as a multiple antigenic peptide (MAP)17,18 to the N-terminal extensions of the two structurally-related peptides. Utilizing a double immunolabeling protocol for antisera generated in the same host species, we mapped the neural distribution of the neuropeptides. We observed that DPKQDFMRFamide and SDNFMRFamide have distinct, non-overlapping cellular expression patterns suggesting that the precursor is differentially processed. Thus, our data support the conclusion that Drosophila contains cell-specific proteolytic enzymes to posttranslationally cleave a polypeptide protein precursor producing structurally-related neuropeptides that may be functionally dissimilar.
MATERIALS AND METHODS Synthesis and characterization of antigens The antigens, DPKQD-MAP and SDNFM-MAP, were each synthesized as a multiple antigenic peptide (MAP), a core matrix of branching lysine onto which the peptide was attached through the carboxyl terminus at eight branch sites.17,18 Antisera production Antisera were raised in New Zealand white rabbits in accordance with institutional guidelines. Initial immunizations were subcutaneous injections at multiple sites of a total of 1 m/l of antigen emulsified in Freund’s complete adjuvant. Subsequent boosts were given every week by subcutaneous injections of 0.1–0.5 m/l of antigen in Freund’s incomplete adjuvant. Immunocytochemical grade FMRFamide antisera raised in rabbits were purchased from Peninsula Labs. Characterization of antisera Antisera were purified on peptide affinity columns made by coupling the appropriate antigen to Affi-gel 10 resin (Bio-Rad Labs) as previously described.19–21 Incubating antisera raised to DPKQD-MAP with DPKQDFMRFamide or DPKQD-MAP abolished all staining, while incubation with FMRFamide did not. Incubating antisera raised to SDNFM-MAP with SDNFM-MAP abolished all staining, while incubating with FMRFamide did not. Immunofluorescence protocol Indirect immunofluorescence was done as previously described.19 Whole mount tissue preparations of central nervous system tissue were prepared from third instar larvae. Tissue was dissected in cold, calcium-free Neuropeptides (1999) 33(1), 35–40
Drosophila Ringer’s solution (130 mM NaCl, 4.7 mM KCl, 1.8 mM MgCl2, 0.74 mM Na2H2PO4, pH 7), fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 4 h, rinsed for 1 h with 2–3 changes of PTN (0.1 M sodium phosphate, pH 7.4, 0.3% Triton X-100, 0.2% NaN3 with 0.1% BSA), and incubated in primary antisera overnight at 4°C. Tissue was rinsed for 1 h with 3–4 changes of PTN, incubated for 4–6 h in goat anti-rabbit secondary antibody, and rinsed for 3–4 h with 8–12 changes of PTN. Tissue was then rinsed for 30 min in 4 mM sodium carbonate, pH 9.5, and mounted in 80% glycerol in 100 mM sodium carbonate, pH 9.5, containing 5% n-propyl gallate for microscopy. Double-label immunofluorescence was done as described previously with the following modifications. After incubation with the first primary antisera and goat anti-rabbit Cy3-labeled Fab fragment (Jackson ImmunoResearch Labs), the tissue was rinsed in PTN for 2 h, and incubated in FITC-conjugated goat anti-rabbit secondary antibody (Sigma Chemical Co.) for 4–6 h. Tissue was then washed extensively and prepared for microscopy as described above. Microscopy and data collection Fluorescent signal was imaged with a Bio-Rad MRC600 Iaser scanning confocal microscope equipped with a Kr-Ar laser attached to a Nikon inverted microscope. Optical sections or z series were collected using the Comos program. Adobe photoshop software, version 3.0, was used to process the data and images were printed with a Kodak XLS8600 printer. The terminology used to identify the antisera stained cells is consistent with previous publications.19,22–24 Immunoreactivity was observed bilaterally symmetric to the midline such that reference to one cell indicates the presence of a pair of cells positioned bilaterally symmetric to one another. Signal intensity was strong and consistent, and eight or more preparations were analyzed for each experiment. Antisera raised to DPKQDMAP and SDNFM-MAP are referred to as DPKQDFMRFamide antisera and SDNFMRFamide antisera, respectively. RESULTS In the larval central nervous system tissue, DPKQDFMRFamide antisera stain two neurons in the superior protocerebrum (SP1 and SP2), two in the subesophageal ganglion (SE2 and Sv), one in each of the three thoracic ganglion (T1–3), one medial neuron in the second thoracic ganglion (T2dm), and one in an abdominal ganglion (A8). (Fig. 1) Immunoreactive processes from SP1 project to the medial commissure of the brain lobes. © 1999 Harcourt Brace & Co. Ltd
Antisera to multiple antigenic peptides 37
Fig. 1 DPKQDFMRFamide immunoreactive material in the larval central nervous system. Antisera to DPKQD-MAP stain cells in the superior protocerebrum (SP1 and SP2), the subesophageal ganglion (Sv and SE2), the thoracic ganglia (T1-3 and T2dm), and an abdominal ganglion (A8). The bar in the lower right-hand corner represents 50 microns.
Fig. 2 SDNFMRFamide immunoreactive material in the larval central nervous system. Antisera to SDNFM-MAP stain cells in the subesophageal ganglion (SE). The bar in the lower right-hand corner represents 50 microns.
Immunoreactive processes project from SE2 along the midline of the ventral ganglion. T1–3 each has an immunoreactive process that projects medially toward the midline. No immunoreactive process was observed from SP2, Sv, T2dm, or A8. Based on position and number of cells, DPKQDFMRFamide immunoreactive material is present in neurons that contain the FMRFamide precursor.24 SDNFMRFamide antisera stain several neurons in the subesophageal ganglion (SE) (Fig. 2). Immunoreactive processes originating from SE neurons project anteriorly along the midline to the superior protocerebrum where they extend laterally terminating in numerous varicosities. Processes extend from the SDNFMRFamide antisera stained neurons laterally to the subesophageal ganglion, then turn and project posteriorly to the ventral ganglion along the midline. Immunoreactive processes from SDNFMRFamide antisera stained neurons also extend laterally and anteriorly to the region near the esophagus.
Based on position and number of cells, SDNFMRFamide immunoreactive material is present in neurons that contain the FMRFamide precursor.24 DPKQDFMRFamide antisera stain a subset of neurons recognized by FMRFamide antisera (Fig. 3). DPKQDFMRFamide and FMRFamide immunoreactivity are present in SP1 and SP2 protocerebral neurons, SE2 and Sv subesophageal neurons, T1–3 and T2dm thoracic neurons, and the A8 abdominal neuron. SDNFMRFamide antisera also stain a subset of neurons recognized by FMRFamide antisera (Fig. 4). SDNFMRFamide and FMRFamide immunoreactivity are present in the SE subesophageal neurons. Double immunolabeling was done with DPKQDFMRFamide antisera and SDNFMRFamide antisera in the same tissue preparation (Fig. 5). Our results indicate that DPKQDFMRFamide and SDNFMRFamide expression patterns do not overlap. The two subesophageal neurons stained by DPKQDFMRFamide
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38 Nichols et al.
Fig. 3 DPKQDFMRFamide and FMRFamide immunoreactive materials in the larval central nervous system. A red fluorescent signal is the result of the Cy3-labeled secondary antibody used to recognize DPKQDFMRFamide antisera and a green fluorescent signal reflects the FITC-labeled second antibody used to recognize FMRFamide antisera. The yellow fluorescent signal indicates that both DPKQDFMRFamide and FMRFamide immunoreactive materials are present. The bar in the lower right-hand corner represent 50 microns.
Fig. 4 SDNFMRFamide and FMRFamide immunoreactive materials in the larval central nervous system. A red fluorescent signal is the result of the Cy3-labeled secondary antibody used to recognize SDNFMRFamide antisera and a green fluorescent signal reflects the FITC-labeled secondary antibody used to recognize FMRFamide antisera. The yellow fluorescent signal indicated that both SDNFMRFamide and FMRFamide immunoreactive materials are present. The bar in the lower right-hand corner represents 50 microns.
antisera are posterior to the cluster of neurons recognized by SDNFMRFamide antisera. The DPKQDFMRFamide-immunoreactive process from the subesophageal neuron that extends into the ventral ganglion along the midline is medial to the SDNFMRFamide-immunoreactive process (Fig. 5).
diverse biological activities. We are interested in regulation of expression of FMRFamide-related peptides. In this manuscript we report raising antisera to DPKQDFMRFamide and SDNFMRFamide and determining the distribution of the structurally-related neuropeptides. Double immunolabeling is critical for simultaneously visualizing two antigens in the same tissue preparation. Methods using antisera from the same species29–34 have general application, but can have drawbacks. Some protocols rely on preabsorption with normal serum to help avoid crossreactivity, requiring an additional procedural step with possible unsatisfactory results. Other procedures utilize enzymatic or chemical reactions that can result in high background. Linking a fluorescent or chemical tag to primary antisera requires manipulation of the antisera. We conducted a double labeling protocol using antisera raised to MAP antigens in different
DISCUSSION Regulation of neuropeptide signaling is critical to physiology, hence, several mechanisms exist for modulating neurotransmission. One mode of regulation is at the posttranslational level with cell-specific proteolytic processing enzymes cleaving a precursor to produce neuropeptides in unique expression patterns.25–28. Through differential processing structurally-related peptides present in the same precursor can have different cellular or developmental distribution patterns and Neuropeptides (1999) 33(1), 35–40
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Antisera to multiple antigenic peptides 39
structurally-related peptides encoded in the FMRFamide gene. Peptide-specific antisera that distinguish between structurally-related neuropeptides are crucial for establishing cellular distribution and identifying sites of action to decipher gene regulation and elucidate physiological function. In this study we determined that DPKQDFMRFamide antisera and SDNFMRFamide antisera stain a distinct pattern of cells and, thus, are not co-expressed. In addition, we show that DPKQDFMRFamide and SDNFMRFamide immunoreactivities are present in cells also stained by antisera to FMRFamide. The neurons stained by DPKQDFMRFamide antisera and SDNFMRFamide antisera are similar in distribution and number with those previously identified as containing proFMRFamide.24 Thus, these data suggest that the Drosophila FMRFamide gene undergoes differential processing to produce unique distributions of structurally similar peptides with different activities. ACKNOWLEDGEMENT Research grants from The National Science Foundation and The American Heart Association Michigan Affiliate to RN are acknowledged. REFERENCES Fig. 5 SDNFMRFamide and DPKQDFMRFamide immunoreactive materials in the larval central nervous system. Red fluorescent signal reflects the Cy3-labeled secondary antibody used to recognize SDNFMRFamide antisera and the green fluorescent signal reflects the FITC-labeled secondary antibody used to recognize DPKQDFMRFamide antisera. The absence of any yellow fluorescent signal indicates that DPKQDFMRFamide and SDNFMRFamide immunoreactive materials are not present in the same cells or processes. The bar in the lower right-hand corner represents 50 microns.
animals of the same host species. The first primary antisera applied are detected by fluorescently-labeled Fab fragment to prevent the binding of the second primary antisera to the first secondary antibody. The use of a MAP can be advantageous since it can be designed to a short length of sequence. Neuropeptides are frequently short in length and, if there is structure similarity, only a few amino acid residues may distinguish between peptides. Also, the use of a MAP does not require a carrier molecule which itself may be antigenic.17,18 Antisera to FMRFamide stain numerous cells in Drosophila neural tissue 11,12,22, however, because FMRFamide antisera recognize a common structure in numerous peptides, these data cannot be unambiguously interpreted. We raised antisera that distinguish between DPKQDFMRFamide and SDNFMRFamide, © 1999 Harcourt Brace & Co. Ltd
1. Raffa RB. The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals. Peptides 1988; 9: 915–922. 2. Price DA, Greenberg MJ. The hunting of the FaRPs: the distribution of FMRFamide-related peptides. Biol Bull 1989; 177: 198–205. 3. Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977; 197: 670–672. 4. Marks NJ, Shaw C, Maule AG et al. Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the presence of more than one FMRFamide-related peptideencoding gene. Biochem Biophys Res Commun 1995; 217: 845–851. 5. Price DA, Doble KE, Lesser W et al. The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current. Biol Bull 1996; 191: 341–352. 6. Edison AS, Messinger LA, Stretton AO. afp-1: a gene encoding multiple transcripts of a new class of FMRFamide-like neuropeptides in the nematode Ascaris suum Peptides 1997; 18: 929–935. 7. Nelson LS, Kim K, Memmott JE, Li C. FMRFamide-related gene family in the nematode, Caenorhabditis elegans. Mol Brain Res 1998; 58: 103–111. 8. Nichols R, Schneuwly SA, Dixon JE. Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. J Biol Chem 1988; 263: 12167–12170. 9. Nichols R. Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol Cell Neurosci 1992; 3: 342–347. 10. Nichols R. Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides. J Mol Neurosci 1992; 3: 213–218.
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