- Email: [email protected]
Multiple Antigenic Peptides Designed to Structurally Related Drosophila Peptides1
Peptides, Vol. 18, No. 1, pp. 41–45, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(96)00279-3
Multiple Antigenic Peptides Designed to Structurally Related Drosophila Peptides1 R. NICHOLS,*†2 J. McCORMICK† AND I. LIM† Departments of *Biological Chemistry and †Biology, 830 N. University St., University of Michigan, Ann Arbor, MI 48109-1048 Received 1 May 1996; Accepted 17 October 1996 NICHOLS, R., J. McCORMICK AND I. LIM. Multiple antigenic peptides designed to structurally related Drosophila peptides. PEPTIDES 18(1) 41–45, 1997.—We have isolated TDVDHVFLRFamide (DMS), FDDYGHMRFamide (DSK), and DPKQDFMRFamide from Drosophila melanogaster. These peptides, structurally related by a common C-terminus -XRFamide, where X Å L or M, are encoded by three different genes. To determine cellular expression, we have generated antisera to multiple antigenic peptides and performed double-label immunofluorescence using antisera raised in the same species host animal. Our results indicate that DMS and DSK immunoreactive materials have unique, non-overlapping expression patterns, while DMS and DPKQDFMRFamide immunoreactive materials colocalize in two superior protocerebrum neurons, and DSK and DPKQDFMRFamide immunoreactive materials colocalize in one superior protocerebrum neuron, one subesophageal ganglion neuron, and three thoracic ganglia neurons. q 1997 Elsevier Science Inc. Double-label immunofluorescence
Dromyosuppressin
Drosulfakinin
PEPTIDES present in neural tissue can often be grouped into families based on a similar chemical structure. Because of structural similarity, it is important to consider the presence of structurally-related neuropeptides when designing antigens and interpreting immunolocalization data. One neuropeptide family that has the common structure -XRFamide, where X Å L or M, is represented by the tetrapeptide PheMetArgPheNH2 ( FMRFamide ) , a cardioexcitatory peptide isolated from mollusc ( 10 ) . FMRFamide-related materials serve as hormones and transmitters in various actions in both invertebrates and vertebrates ( 11, 12 ) . Frequently a single organism contains multiple FMRFamide-like peptides. In Drosophila melanogaster , the myosuppressin gene ( Dms ) encodes TDVDHVFLRFamide ( dromyosuppressin or DMS ) , the sulfakinin gene ( Dsk ) encodes FDDYGHMRFamide, where Y represents a sulfated tyrosyl residue ( drosulfakinin or DSK I ) , and the FMRFamide gene encodes DPKQDFMRFamide (3–6, 13). To study the cellular distribution of Drosophila melanogaster FMRFamide-like peptides, we have generated antisera specific to TDVDHVFLRFamide, FDDYGHMRFamide, and DPKQDFMRFamide. Multiple antigenic peptide ( MAP ) antigens ( 9 ) were designed to the unique N-terminal structures of the peptides and antisera raised in rabbits. Double-label immunofluorescence data indicate that DMS, DSK, and DPKQDFMRFamide immunoreactive materials are expressed in several neurons throughout the brain and ventral
FMRFamide
Invertebrate neuropeptides
ganglion. In general, neurons and processes are stained by only one of the antisera, for example, the cellular expression patterns of DMS and DSK immunoreactive materials are unique and non-overlapping indicating that DMS and DSK are not expressed in the same neurons. In contrast, the individual staining patterns of DMS and DSK do overlap to some extent with the staining pattern of DPKQDFMRFamide providing evidence for the coexistence of structurally-related neuropeptides in the Drosophila melanogaster central nervous system. MATERIALS AND METHODS
Synthesis and Characterization of Antigens The antigens, FDDYGH-MAP, TDVDHV-MAP, and DPKQD-MAP, where MAP represents multiple antigenic peptide (9), were synthesized with a Rainin Symphony multiple peptide synthesizer. The procedure for synthesis on the lysyl core followed standard operating conditions (9). Antisera Production Antisera were raised in New Zealand white rabbits using two animals per antigen with initial immunizations by subcutaneous injections at multiple sites of a total of 0.5 milligram of antigen emulsified in Freund’s complete adjuvant. Subsequent boosts were given every week by subcutaneous injections of 0.25 mil-
1 Taken in part from a paper presented at a satellite symposium on Insect Neuropeptides during the Seventh Annual Neuropeptide Conference, February 1–6, 1996, Breckenridge, CO. 2 Requests for reprints should be addressed to R. Nichols, 830 N. University St., University of Michigan, Ann Arbor, MI 48109-1048; Fax: 313747-0884; E-Mail: [email protected].
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ligrams of antigen in Freund’s incomplete adjuvant. Antisera titers were analyzed by indirect immunofluorescence of whole mount third instar larval central nervous system tissue as previously described (2, 14). Purification of Antisera. To purify antisera, peptide affinity columns were made by coupling the appropriate antigen to Affi-gel 10 resin (Bio-Rad Labs) in dry dimethyl sulfoxide and 1% triethylamine according to the manufacturer’s instructions. The amount of antigen coupled to the resin was 2 milligrams peptide/milliliter resin. Each column, containing approximately 5 milliliters of resin, was equilibrated in 5 1 PBS (680mM NaCl, 25mM Na2HPO4 , 9mM KH2PO4 , 13mM KCl, pH 7) by rinsing with 5 column volumes of 5 1 PBS. Column chromatography was done at 47C with a flow rate of approximately 10 milliliters per hour. The flow through was collected and reapplied to the column twice after which the column was washed with 10–20 column volumes of 5 1 PBS to reduce non-specific binding. The bound antisera were eluted with application of 10 milliliters of 0.1M sodium citrate, pH 2.5, and then 10 milliliters 0.1M triethylamine, pH 11.5, collecting directly into 20 milliliters of 1M Tris, pH 8, with stirring. For subsequent use, the column was regenerated with 10 column volumes of 6M guanidine HCl, rinsed with 2 column volumes of 5 1 PBS, and stored at 47C in 5 1 PBS with 0.2% sodium azide. The eluted antisera were dialyzed against 4 liters of 0.01 1 PBS at 47C for 24 h with 4 changes of buffer. The dialyzed antisera were aliquoted, lyophilized, and stored dry at 0207C. For experimental use, an aliquot was resuspended in sterile water to yield antisera in 1 1 PBS. Characterization of Antisera Antisera specificity was determined by the choice of the antigen and affinity purification, as well as preincubation studies. Affinity-purified antisera were further characterized by incubation of the antisera with peptides prior to immunolocalization. Control experiments included antisera preincubated with FDDYGHMRFamide, TDVDHVFLRFamide, and DPKQDFMRFamide at 0.0001M prior to use in immunofluorescence. Indirect Immunofluorescent Protocol Tissue was dissected from third instar larvae and double-label immunofluorescence performed. The protocol was a modification of a previously reported single-label method (2). After incubation with the first primary antisera for 4–6 h and CY3-conjugated Fab fragment goat anti-rabbit secondary antibody (Jackson ImmunoResearch Labs) for 4-6 h, tissue was rinsed in 0.5 M sodium phosphate, pH 7.2, containing 0.2% Triton X-100 and 1% sodium azide (PTN) for 2 h, incubated in the second primary antisera for 4–6 h, rinsed in PTN for 2 h, and incubated in FITCconjugated goat anti-rabbit secondary antibody (Sigma) for 4-6 h. Tissue was then washed and prepared for microscopy as previously described (2). Microscopy and Data Analysis Fluorescence was imaged with a Bio-Rad MRC600 laser scanning confocal microscope equipped with a Kr-Ar laser attached to a Nikon inverted microscope at wavelengths corresponding to CY3 and FITC. Data, collected at 400 1 magnification, were processed with Adobe Photoshop and transferred to Kodak Ektachrome 100 plus color reversal slide film using a Macintosh Quadra 800 and Lasergraphics LFR-X.
RESULTS
The nomenclature used in describing cellular expression is based on previous publications identifying FMRFamide-like immunoreactive materials (2, 8; see Fig. 1). Signal intensity was strong and consistent and no fewer than eight preparations were analyzed for each experiment. The DMS and DSK staining patterns in larval central nervous system tissue are unique and non-overlapping (Figs. 1a, 2a). DMS antisera, recognized by FITC-labeled secondary antibody (green signal), stained neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DSK antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the superior protocerebrum, the lateral protocerebrum, the medial protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. The superior protocerebrum neurons containing DMS immunoreactive material are positioned posterior or lateral to the neuron stained by DSK antisera. The medial protocerebrum neurons containing DMS immunoreactive material are lateral to those stained by DSK antisera. The subesophageal ganglion neuron containing DMS immunoreactive material is ventral to the neuron stained by DSK antisera. Based on the position of staining, DMS antisera stained SP3 and SP2 neurons and DSK antisera stained the SP1 neuron in the superior protocerebrum. DMS antisera stained the MP2 neurons and DSK antisera stained the MP1 neurons in the medial protocerebrum. DMS antisera stained the SE2v neuron and DSK antisera stained the SE2 neuron in the subesophageal ganglion (8). The DMS and DPKQDFMRFamide staining patterns in larval central nervous system tissue are, for the most part, non-overlapping (Figs. 1b, 2b). DMS antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DPKQDFMRFamide antisera, recognized by FITC-labeled secondary antibody (green signal), stained neurons in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. The medial superior protocerebrum neurons containing DMS immunoreactive material are also stained by DPKQDFMRFamide antisera, while the lateral superior protocerebrum neurons are stained by DMS or DPKQDFMRFamide antisera, but not both. The subesophageal ganglion neuron containing DMS immunoreactive material is ventral to the neuron stained by DPKQDFMRFamide antisera. Based on the position of staining in the superior protocerebrum, DMS and DPKQDFMRFamide antisera recognized the SP3 neurons. DMS antisera stained the SE2v neuron and DPKQDFMRFamide antisera stained the SE2 neuron in the subesophageal ganglion. The DSK and DPKQDFMRFamide staining patterns in larval central nervous system tissue have considerable overlap (Figs. 1c, 2c). DSK antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the lateral protocerebrum and medial protocerebrum, not stained by DPKQDFMRFamide antisera, recognized by FITC-labeled secondary antibody (green signal). However, both DSK and DPKQDFMRFamide antisera stained neurons in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. Based on position, DSK and DPKQDFMRFamide antisera stained the SP1 neuron in the superior protocerebrum, the SE2 neuron in the subesophageal ganglion, and the T1-3 neurons in the thoracic ganglia. DISCUSSION
Multiple peptide messengers can colocalize in a single neuron (1). Raising antisera to a unique sequence contained in a peptide
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FIG. 1. (upper left) Schematic illustrating the pattern of DMS and DSK immunoreactivity and the nomenclature identifying the neurons stained by the antisera. (upper right) Schematic illustrating the pattern of DMS and DPKQDFMRFamide immunoreactivity and the nomenclature identifying the neurons stained by the antisera. (lower left) Schematic illustrating the pattern of DSK and DPKQDFMRFamide immunoreactivity and the nomenclature identifying the neurons stained by the antisera.
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FIG. 2. (a) DMS and DSK immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. FITC-labeled secondary antibody (green signal) recognized DMS antisera. CY3-labeled secondary antibody (red signal) recognized DSK antisera. DMS immunoreactive material is present in neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DSK immunoreactive material is present in the superior protocerebrum, the lateral protocerebrum, the medial protocerebrum, the subesophageal ganglion, and the
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is a simple yet powerful approach to distinguish between structurally-related peptides and address the question of coexistence. In the work described in this manuscript we utilized MAP antigens designed to the unique N-terminal amino acid extensions and generated antisera to identify specific -XRFamide-containing peptides present in the Drosophila melanogaster central nervous system. There are several advantages to using a MAP antigen. When generating antisera to a small molecule it is typically necessary to conjugate it to a larger molecule or carrier which may be antigenic itself. When using a MAP antigen the actual peptide fragment contributes a major share of the mass and structure of the antigen, unlike conjugating a small peptide to a large carrier molecule. In addition, a MAP antigen can be synthesized directly, eliminating the conjugation step. Also, the multiplicity of the peptide in a MAP antigen provides for a highly antigenic molecule. The antisera used in this study were raised in rabbits. Generating antisera in different animals of the same host species can prove advantageous. For example, animal care and housing as well as blood collection and handling techniques may be simpli-
fied. In addition, there may be more fluorescently-labeled secondary antibodies readily available for a host species. The availability of antisera that distinguish between structurally-related peptides is an important experimental tool in studying the expression and function of gene products and the processing of polypeptide precursors. As a result of using antisera that distinguish among Drosophila melanogaster FMRFamidelike peptides, we have determined that DMS and DSK do not colocalize, while DPKQDFMRFamide colocalizes with both DMS and DSK. It is not known why the pattern of expression of DMS and DSK is exclusive, nor why there is extensive overlap of the DPKQDFMRFamide and DSK staining patterns. The expression patterns may reflect a similarity in sites of action or regulatory or modulatory role of these peptides. ACKNOWLEDGMENTS
All peptides were synthesized and characterized at The University of Michigan Protein Structure Facility, P.C. Andrews, Ph.D., Director. We thank Professor Andrews for suggesting the use of multiple antigenic peptides. This work was supported by NSF IBN #9409623 to RN.
REFERENCES 1. Ho¨kfelt, T.; Holets, V. R.; Staines, W.; Meister, B.; Melander, T.; Schalling, M.; Schultzberg, M.; Freedman, J.; Bjo¨rklund, H.; Olson, L.; Lindh, B.; Elfvin, L-G.; Lundberg, J. M.; Lindgren, J. A.; Samuelsson, B.; Pernow, B.; Terenius, L.; Post, C.; Everitt, B.; Goldstein, M. In: Ho¨kfelt, T.; Fuxe, K.; Pernow, B. Eds. Progress in brain research, vol. 68. Coexistence of neuronal messengers: A new principle in chemical transmission. 1986:33–70. 2. McCormick, J.; Nichols, R. Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster. J. Comp. Neurol. 338:279; 1993. 3. Nambu, J. R.; Murphy–Erdosh, C.; Andrews, P. C.; Feistner, G. J.; Scheller, R. H. Isolation and characterization of a Drosophila neuropeptide gene. Neuron 1:55; 1988. 4. Nichols, R.; Schneuwly, S. A.; Dixon, J. E. Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. J. Biol. Chem. 263:12167; 1988. 5. Nichols, R. Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides. J. Mol. Neurosci. 3:213; 1992. 6. Nichols, R. Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol. Cell. Neurosci. 3:342; 1992. 7. Nichols, R.; McCormick, J.; Lim, I.; Caserta, L. Cellular expression of the Drosophila melanogaster FMRFamide neuropeptide gene
8. 9.
10. 11. 12. 13.
14.
product DPKQDFMRFamide: Evidence for differential processing of the FMRFamide polypeptide precursor. J. Mol. Neurosci. 6:1; 1995. Nichols, R.; Lim, I. Spatial and temporal expression pattern of the drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system. Cell Tiss. Res. 282:107; 1996. Posnett, D. N.; Tam, J. P. In: Langone, J. J., Ed. Methods in enzymology, vol. 179. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. New York: Academic Press, 1989:739–746. Price, D. A.; Greenberg, M. J. Structure of a molluscan cardioexcitatory neuropeptide. Science 197:670; 1977. Price, D. A.; Greenberg, M. J. The hunting of the FaRPs: The distribution of FMRFamide-related peptides. Biol. Bull. 177:198; 1989. Raffa, R. B. The action of FMRFamide (Phe-Met-Arg-Phe-NH2 ) and related peptides on mammals. Peptides 9:915; 1988. Schneider, L. E.; Taghert, P. H. Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to PheMet-Arg-Phe-NH2 (FMRFamide). Proc. Natl. Acad. Sci. 85:1993; 1988. White, K.; Hurteau, P.; Punsal, P. Neuropeptide-FMRFamide-like immunoreactivity in Drosophila: Development and distribution. J. Comp. Neurol. 247:430; 1986.
ventral ganglion. A yellow signal appears in the superior protocerebrum of the right brain lobe because the two neurons stained individually by DMS and by DSK antisera were positioned such that the fluorescence overlapped. However, the neurons are in different sections of the whole mount preparation; the DMS-stained neuron is dorsal to the DSK-stained neuron. This difference of position can be observed for the corresponding neuron in the left brain lobe. (The line in the lower right corner represents 50 microns. (b) DMS and DPKQDFMRFamide immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. CY3-labeled secondary antibody (red signal) recognized DMS antisera. FITClabeled second antibody (green signal) recognized DPKQDFMRFamide antisera. DMS immunoreactive material is present in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DPKQDFMRFamide immunoreactive material is present in the superior protocerebrum, the subesophageal ganglion, the second thoracic ganglion, and the three thoracic ganglia. DMS and DPKQDFMRFamide antisera both stain the two superior protocerebrum neurons (yellow signal), while the subesophageal ganglion neuron stained by DMS antisera is ventral to the neuron stained by DPKQDFMRFamide antisera. A yellow signal appears in the subesophageal ganglion, left of midline, because the two neurons stained individually by DMS and by DPKQDFMRFamide antisera were positioned such that the fluorescence overlapped. The difference in position can be observed for the corresponding neuron right of the midline. (The line in the lower right corner represents 50 microns.) (c) DSK and DPKQDFMRFamide immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. CY3-labeled secondary antibody (red signal) recognized DSK antisera. FITC-labeled secondary antibody (green signal) recognized DPKQDFMRFamide antisera. DSK immunoreactive material is present in neurons in the lateral protocerebrum and the medial protocerebrum, neurons not stained by DPKQDFMRFamide antisera. DPKQDFMRFamide immunoreactive material is present in neurons in the superior protocerebrum and the second thoracic ganglion, neurons not stained by DSK antisera. DSK and DPKQDFMRFamide immunoreactive materials colocalize in neurons present in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. (The line in the lower right corner represents 50 microns.)
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Multiple Antigenic Peptides Designed to Structurally Related Drosophila Peptides1 R. NICHOLS,*†2 J. McCORMICK† AND I. LIM† Departments of *Biological Chemistry and †Biology, 830 N. University St., University of Michigan, Ann Arbor, MI 48109-1048 Received 1 May 1996; Accepted 17 October 1996 NICHOLS, R., J. McCORMICK AND I. LIM. Multiple antigenic peptides designed to structurally related Drosophila peptides. PEPTIDES 18(1) 41–45, 1997.—We have isolated TDVDHVFLRFamide (DMS), FDDYGHMRFamide (DSK), and DPKQDFMRFamide from Drosophila melanogaster. These peptides, structurally related by a common C-terminus -XRFamide, where X Å L or M, are encoded by three different genes. To determine cellular expression, we have generated antisera to multiple antigenic peptides and performed double-label immunofluorescence using antisera raised in the same species host animal. Our results indicate that DMS and DSK immunoreactive materials have unique, non-overlapping expression patterns, while DMS and DPKQDFMRFamide immunoreactive materials colocalize in two superior protocerebrum neurons, and DSK and DPKQDFMRFamide immunoreactive materials colocalize in one superior protocerebrum neuron, one subesophageal ganglion neuron, and three thoracic ganglia neurons. q 1997 Elsevier Science Inc. Double-label immunofluorescence
Dromyosuppressin
Drosulfakinin
PEPTIDES present in neural tissue can often be grouped into families based on a similar chemical structure. Because of structural similarity, it is important to consider the presence of structurally-related neuropeptides when designing antigens and interpreting immunolocalization data. One neuropeptide family that has the common structure -XRFamide, where X Å L or M, is represented by the tetrapeptide PheMetArgPheNH2 ( FMRFamide ) , a cardioexcitatory peptide isolated from mollusc ( 10 ) . FMRFamide-related materials serve as hormones and transmitters in various actions in both invertebrates and vertebrates ( 11, 12 ) . Frequently a single organism contains multiple FMRFamide-like peptides. In Drosophila melanogaster , the myosuppressin gene ( Dms ) encodes TDVDHVFLRFamide ( dromyosuppressin or DMS ) , the sulfakinin gene ( Dsk ) encodes FDDYGHMRFamide, where Y represents a sulfated tyrosyl residue ( drosulfakinin or DSK I ) , and the FMRFamide gene encodes DPKQDFMRFamide (3–6, 13). To study the cellular distribution of Drosophila melanogaster FMRFamide-like peptides, we have generated antisera specific to TDVDHVFLRFamide, FDDYGHMRFamide, and DPKQDFMRFamide. Multiple antigenic peptide ( MAP ) antigens ( 9 ) were designed to the unique N-terminal structures of the peptides and antisera raised in rabbits. Double-label immunofluorescence data indicate that DMS, DSK, and DPKQDFMRFamide immunoreactive materials are expressed in several neurons throughout the brain and ventral
FMRFamide
Invertebrate neuropeptides
ganglion. In general, neurons and processes are stained by only one of the antisera, for example, the cellular expression patterns of DMS and DSK immunoreactive materials are unique and non-overlapping indicating that DMS and DSK are not expressed in the same neurons. In contrast, the individual staining patterns of DMS and DSK do overlap to some extent with the staining pattern of DPKQDFMRFamide providing evidence for the coexistence of structurally-related neuropeptides in the Drosophila melanogaster central nervous system. MATERIALS AND METHODS
Synthesis and Characterization of Antigens The antigens, FDDYGH-MAP, TDVDHV-MAP, and DPKQD-MAP, where MAP represents multiple antigenic peptide (9), were synthesized with a Rainin Symphony multiple peptide synthesizer. The procedure for synthesis on the lysyl core followed standard operating conditions (9). Antisera Production Antisera were raised in New Zealand white rabbits using two animals per antigen with initial immunizations by subcutaneous injections at multiple sites of a total of 0.5 milligram of antigen emulsified in Freund’s complete adjuvant. Subsequent boosts were given every week by subcutaneous injections of 0.25 mil-
1 Taken in part from a paper presented at a satellite symposium on Insect Neuropeptides during the Seventh Annual Neuropeptide Conference, February 1–6, 1996, Breckenridge, CO. 2 Requests for reprints should be addressed to R. Nichols, 830 N. University St., University of Michigan, Ann Arbor, MI 48109-1048; Fax: 313747-0884; E-Mail: [email protected].
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ligrams of antigen in Freund’s incomplete adjuvant. Antisera titers were analyzed by indirect immunofluorescence of whole mount third instar larval central nervous system tissue as previously described (2, 14). Purification of Antisera. To purify antisera, peptide affinity columns were made by coupling the appropriate antigen to Affi-gel 10 resin (Bio-Rad Labs) in dry dimethyl sulfoxide and 1% triethylamine according to the manufacturer’s instructions. The amount of antigen coupled to the resin was 2 milligrams peptide/milliliter resin. Each column, containing approximately 5 milliliters of resin, was equilibrated in 5 1 PBS (680mM NaCl, 25mM Na2HPO4 , 9mM KH2PO4 , 13mM KCl, pH 7) by rinsing with 5 column volumes of 5 1 PBS. Column chromatography was done at 47C with a flow rate of approximately 10 milliliters per hour. The flow through was collected and reapplied to the column twice after which the column was washed with 10–20 column volumes of 5 1 PBS to reduce non-specific binding. The bound antisera were eluted with application of 10 milliliters of 0.1M sodium citrate, pH 2.5, and then 10 milliliters 0.1M triethylamine, pH 11.5, collecting directly into 20 milliliters of 1M Tris, pH 8, with stirring. For subsequent use, the column was regenerated with 10 column volumes of 6M guanidine HCl, rinsed with 2 column volumes of 5 1 PBS, and stored at 47C in 5 1 PBS with 0.2% sodium azide. The eluted antisera were dialyzed against 4 liters of 0.01 1 PBS at 47C for 24 h with 4 changes of buffer. The dialyzed antisera were aliquoted, lyophilized, and stored dry at 0207C. For experimental use, an aliquot was resuspended in sterile water to yield antisera in 1 1 PBS. Characterization of Antisera Antisera specificity was determined by the choice of the antigen and affinity purification, as well as preincubation studies. Affinity-purified antisera were further characterized by incubation of the antisera with peptides prior to immunolocalization. Control experiments included antisera preincubated with FDDYGHMRFamide, TDVDHVFLRFamide, and DPKQDFMRFamide at 0.0001M prior to use in immunofluorescence. Indirect Immunofluorescent Protocol Tissue was dissected from third instar larvae and double-label immunofluorescence performed. The protocol was a modification of a previously reported single-label method (2). After incubation with the first primary antisera for 4–6 h and CY3-conjugated Fab fragment goat anti-rabbit secondary antibody (Jackson ImmunoResearch Labs) for 4-6 h, tissue was rinsed in 0.5 M sodium phosphate, pH 7.2, containing 0.2% Triton X-100 and 1% sodium azide (PTN) for 2 h, incubated in the second primary antisera for 4–6 h, rinsed in PTN for 2 h, and incubated in FITCconjugated goat anti-rabbit secondary antibody (Sigma) for 4-6 h. Tissue was then washed and prepared for microscopy as previously described (2). Microscopy and Data Analysis Fluorescence was imaged with a Bio-Rad MRC600 laser scanning confocal microscope equipped with a Kr-Ar laser attached to a Nikon inverted microscope at wavelengths corresponding to CY3 and FITC. Data, collected at 400 1 magnification, were processed with Adobe Photoshop and transferred to Kodak Ektachrome 100 plus color reversal slide film using a Macintosh Quadra 800 and Lasergraphics LFR-X.
RESULTS
The nomenclature used in describing cellular expression is based on previous publications identifying FMRFamide-like immunoreactive materials (2, 8; see Fig. 1). Signal intensity was strong and consistent and no fewer than eight preparations were analyzed for each experiment. The DMS and DSK staining patterns in larval central nervous system tissue are unique and non-overlapping (Figs. 1a, 2a). DMS antisera, recognized by FITC-labeled secondary antibody (green signal), stained neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DSK antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the superior protocerebrum, the lateral protocerebrum, the medial protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. The superior protocerebrum neurons containing DMS immunoreactive material are positioned posterior or lateral to the neuron stained by DSK antisera. The medial protocerebrum neurons containing DMS immunoreactive material are lateral to those stained by DSK antisera. The subesophageal ganglion neuron containing DMS immunoreactive material is ventral to the neuron stained by DSK antisera. Based on the position of staining, DMS antisera stained SP3 and SP2 neurons and DSK antisera stained the SP1 neuron in the superior protocerebrum. DMS antisera stained the MP2 neurons and DSK antisera stained the MP1 neurons in the medial protocerebrum. DMS antisera stained the SE2v neuron and DSK antisera stained the SE2 neuron in the subesophageal ganglion (8). The DMS and DPKQDFMRFamide staining patterns in larval central nervous system tissue are, for the most part, non-overlapping (Figs. 1b, 2b). DMS antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DPKQDFMRFamide antisera, recognized by FITC-labeled secondary antibody (green signal), stained neurons in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. The medial superior protocerebrum neurons containing DMS immunoreactive material are also stained by DPKQDFMRFamide antisera, while the lateral superior protocerebrum neurons are stained by DMS or DPKQDFMRFamide antisera, but not both. The subesophageal ganglion neuron containing DMS immunoreactive material is ventral to the neuron stained by DPKQDFMRFamide antisera. Based on the position of staining in the superior protocerebrum, DMS and DPKQDFMRFamide antisera recognized the SP3 neurons. DMS antisera stained the SE2v neuron and DPKQDFMRFamide antisera stained the SE2 neuron in the subesophageal ganglion. The DSK and DPKQDFMRFamide staining patterns in larval central nervous system tissue have considerable overlap (Figs. 1c, 2c). DSK antisera, recognized by CY3-labeled secondary antibody (red signal), stained neurons in the lateral protocerebrum and medial protocerebrum, not stained by DPKQDFMRFamide antisera, recognized by FITC-labeled secondary antibody (green signal). However, both DSK and DPKQDFMRFamide antisera stained neurons in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. Based on position, DSK and DPKQDFMRFamide antisera stained the SP1 neuron in the superior protocerebrum, the SE2 neuron in the subesophageal ganglion, and the T1-3 neurons in the thoracic ganglia. DISCUSSION
Multiple peptide messengers can colocalize in a single neuron (1). Raising antisera to a unique sequence contained in a peptide
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FIG. 1. (upper left) Schematic illustrating the pattern of DMS and DSK immunoreactivity and the nomenclature identifying the neurons stained by the antisera. (upper right) Schematic illustrating the pattern of DMS and DPKQDFMRFamide immunoreactivity and the nomenclature identifying the neurons stained by the antisera. (lower left) Schematic illustrating the pattern of DSK and DPKQDFMRFamide immunoreactivity and the nomenclature identifying the neurons stained by the antisera.
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FIG. 2. (a) DMS and DSK immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. FITC-labeled secondary antibody (green signal) recognized DMS antisera. CY3-labeled secondary antibody (red signal) recognized DSK antisera. DMS immunoreactive material is present in neurons in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DSK immunoreactive material is present in the superior protocerebrum, the lateral protocerebrum, the medial protocerebrum, the subesophageal ganglion, and the
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EXPRESSION OF DROSOPHILA NEUROPEPTIDES
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is a simple yet powerful approach to distinguish between structurally-related peptides and address the question of coexistence. In the work described in this manuscript we utilized MAP antigens designed to the unique N-terminal amino acid extensions and generated antisera to identify specific -XRFamide-containing peptides present in the Drosophila melanogaster central nervous system. There are several advantages to using a MAP antigen. When generating antisera to a small molecule it is typically necessary to conjugate it to a larger molecule or carrier which may be antigenic itself. When using a MAP antigen the actual peptide fragment contributes a major share of the mass and structure of the antigen, unlike conjugating a small peptide to a large carrier molecule. In addition, a MAP antigen can be synthesized directly, eliminating the conjugation step. Also, the multiplicity of the peptide in a MAP antigen provides for a highly antigenic molecule. The antisera used in this study were raised in rabbits. Generating antisera in different animals of the same host species can prove advantageous. For example, animal care and housing as well as blood collection and handling techniques may be simpli-
fied. In addition, there may be more fluorescently-labeled secondary antibodies readily available for a host species. The availability of antisera that distinguish between structurally-related peptides is an important experimental tool in studying the expression and function of gene products and the processing of polypeptide precursors. As a result of using antisera that distinguish among Drosophila melanogaster FMRFamidelike peptides, we have determined that DMS and DSK do not colocalize, while DPKQDFMRFamide colocalizes with both DMS and DSK. It is not known why the pattern of expression of DMS and DSK is exclusive, nor why there is extensive overlap of the DPKQDFMRFamide and DSK staining patterns. The expression patterns may reflect a similarity in sites of action or regulatory or modulatory role of these peptides. ACKNOWLEDGMENTS
All peptides were synthesized and characterized at The University of Michigan Protein Structure Facility, P.C. Andrews, Ph.D., Director. We thank Professor Andrews for suggesting the use of multiple antigenic peptides. This work was supported by NSF IBN #9409623 to RN.
REFERENCES 1. Ho¨kfelt, T.; Holets, V. R.; Staines, W.; Meister, B.; Melander, T.; Schalling, M.; Schultzberg, M.; Freedman, J.; Bjo¨rklund, H.; Olson, L.; Lindh, B.; Elfvin, L-G.; Lundberg, J. M.; Lindgren, J. A.; Samuelsson, B.; Pernow, B.; Terenius, L.; Post, C.; Everitt, B.; Goldstein, M. In: Ho¨kfelt, T.; Fuxe, K.; Pernow, B. Eds. Progress in brain research, vol. 68. Coexistence of neuronal messengers: A new principle in chemical transmission. 1986:33–70. 2. McCormick, J.; Nichols, R. Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster. J. Comp. Neurol. 338:279; 1993. 3. Nambu, J. R.; Murphy–Erdosh, C.; Andrews, P. C.; Feistner, G. J.; Scheller, R. H. Isolation and characterization of a Drosophila neuropeptide gene. Neuron 1:55; 1988. 4. Nichols, R.; Schneuwly, S. A.; Dixon, J. E. Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. J. Biol. Chem. 263:12167; 1988. 5. Nichols, R. Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides. J. Mol. Neurosci. 3:213; 1992. 6. Nichols, R. Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol. Cell. Neurosci. 3:342; 1992. 7. Nichols, R.; McCormick, J.; Lim, I.; Caserta, L. Cellular expression of the Drosophila melanogaster FMRFamide neuropeptide gene
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product DPKQDFMRFamide: Evidence for differential processing of the FMRFamide polypeptide precursor. J. Mol. Neurosci. 6:1; 1995. Nichols, R.; Lim, I. Spatial and temporal expression pattern of the drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system. Cell Tiss. Res. 282:107; 1996. Posnett, D. N.; Tam, J. P. In: Langone, J. J., Ed. Methods in enzymology, vol. 179. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. New York: Academic Press, 1989:739–746. Price, D. A.; Greenberg, M. J. Structure of a molluscan cardioexcitatory neuropeptide. Science 197:670; 1977. Price, D. A.; Greenberg, M. J. The hunting of the FaRPs: The distribution of FMRFamide-related peptides. Biol. Bull. 177:198; 1989. Raffa, R. B. The action of FMRFamide (Phe-Met-Arg-Phe-NH2 ) and related peptides on mammals. Peptides 9:915; 1988. Schneider, L. E.; Taghert, P. H. Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to PheMet-Arg-Phe-NH2 (FMRFamide). Proc. Natl. Acad. Sci. 85:1993; 1988. White, K.; Hurteau, P.; Punsal, P. Neuropeptide-FMRFamide-like immunoreactivity in Drosophila: Development and distribution. J. Comp. Neurol. 247:430; 1986.
ventral ganglion. A yellow signal appears in the superior protocerebrum of the right brain lobe because the two neurons stained individually by DMS and by DSK antisera were positioned such that the fluorescence overlapped. However, the neurons are in different sections of the whole mount preparation; the DMS-stained neuron is dorsal to the DSK-stained neuron. This difference of position can be observed for the corresponding neuron in the left brain lobe. (The line in the lower right corner represents 50 microns. (b) DMS and DPKQDFMRFamide immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. CY3-labeled secondary antibody (red signal) recognized DMS antisera. FITClabeled second antibody (green signal) recognized DPKQDFMRFamide antisera. DMS immunoreactive material is present in the superior protocerebrum, the medial protocerebrum, and the subesophageal ganglion. DPKQDFMRFamide immunoreactive material is present in the superior protocerebrum, the subesophageal ganglion, the second thoracic ganglion, and the three thoracic ganglia. DMS and DPKQDFMRFamide antisera both stain the two superior protocerebrum neurons (yellow signal), while the subesophageal ganglion neuron stained by DMS antisera is ventral to the neuron stained by DPKQDFMRFamide antisera. A yellow signal appears in the subesophageal ganglion, left of midline, because the two neurons stained individually by DMS and by DPKQDFMRFamide antisera were positioned such that the fluorescence overlapped. The difference in position can be observed for the corresponding neuron right of the midline. (The line in the lower right corner represents 50 microns.) (c) DSK and DPKQDFMRFamide immunoreactivity in larval central nervous system tissue; dorsal view of a whole mount preparation. CY3-labeled secondary antibody (red signal) recognized DSK antisera. FITC-labeled secondary antibody (green signal) recognized DPKQDFMRFamide antisera. DSK immunoreactive material is present in neurons in the lateral protocerebrum and the medial protocerebrum, neurons not stained by DPKQDFMRFamide antisera. DPKQDFMRFamide immunoreactive material is present in neurons in the superior protocerebrum and the second thoracic ganglion, neurons not stained by DSK antisera. DSK and DPKQDFMRFamide immunoreactive materials colocalize in neurons present in the superior protocerebrum, the subesophageal ganglion, and the three thoracic ganglia. (The line in the lower right corner represents 50 microns.)
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