Spatial and temporal analysis of the Drosophila FMRFamide neuropeptide gene product SDNFMRFamide: Evidence for a restricted expression pattern

Neuropeptides (1995) 29, 205-213 © Pearson Professional Ltd 1995

Spatial and Temporal Analysis of the Drosophila FMRFamide Neuropeptide Gene Product SDNFMRFamide: Evidence for a Restricted Expression Pattern R. NICHOLS*, J. B. McCORMICKf, I. A. LIMt and J. S. STARKMANf

Departments of *Biological Chemistry and tBiology, University of Michigan, 4008 Natural Science Building, 830 IV. University St., Ann Arbor, M148109-1048, USA (Reprint requests to RN)

Abstract--The expression of SDNFMRFamide, one of five different FMRFamide-containing peptides encoded by the Drosophila melanogaster FMRFamide gene, has been determined. To study expression, we generated antisera to the N-terminus of SDNFMRFamide to avoid crossreactivity with FMRFamide-containing peptides. The antisera were purified and the specificity characterized. SDNFMRFamide immunoreactive material is present in the central nervous system throughout development. Immunoreactivity is first observed in embryonic neural tissue in a cluster of cells in the subesophageal ganglion and immunoreactive fibers projecting from these cells to the brain and ventral ganglion. This pattern of expression is also observed in neural tissue dissected from larva, pupa, and adult. Double-labelling experiments indicate that cells recognized by SDNFM-antisera are also stained with FMRFamide antisera. Based on position, SDNFMRFamide immunoreactive material is expressed in a limited number of cells that contain the FMRFamide polypeptide precursor. This finding suggests that the Drosophila FMRFamide precursor undergoes differential post-translational processing.

Introduction Several studies have established that peptides present in neural tissue can act as transmitters, modulators, or hormones in important physiological Date received 27 March 1995 Date accepted 30 March 1995 Correspondence to: R. Nichols, 830 N. University St., University of Michigan, Ann Arbor, MI 48109-1048, USA. Tel: 313/7644467, Fax: 313/747-0884, [email protected] (E-mail).

functions. The identification of these neuropeptides and the isolation of their precursors has led to the finding that multiple peptides are often encoded in the same precursor. Frequently, these neuropeptides are synthesized as inactive polypeptide precursors which then undergo post-translational processing and modification events to release multiple bioactive peptides.l'2 The release of peptides from a precursor can be regulated on a developmental or cellular level.

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206 Since the discovery of the tetrapeptide FMRFamide as a molluscan cardio-excitatory peptide in 1977, 3 antisera to FMRFamide have been used to demonstrate that FMRFamide immunoreactive materials are widespread in both invertebrate and vertebrate neural tissue and act as hormones and neurotransmitters of various modulatory actions.4~8 The FMRFamide immunoreactive peptides isolated to date are structurally related by an identical C-terminus -RFamide with different N-terminal amino acid extensions. Three Drosophila meIanogaster genes have been identified that encode -RFamide-containing peptides: dromyosuppressin (Dins), drosulfakinin (Dsk), and FMRFamide. 9-~3 The Drosophila melanogaster FMRFamide gene can be predicted to encode five different-FMRFamide-containing peptides, three of which have been isolated: SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide.I~ Antisera to FMRFamide have been used to identify a set of approximately 50 immunoreactive cells in the Drosophila melanogaster central nervous system and gut. ~4-~8 However, because there are multiple -RFamide-containing peptides in Drosophila and because FMRFamide antisera recognize the common C-terminal -RFamide, these data cannot be interpreted to determine the identify of the FMRFamide-containing peptide(s) present in a cell. An immunocytochemical study done using antisera to the FMRFamide polypeptide precursor defines the distribution of the precursor but does not provide any information on the FMRFamidecontaining peptides processed from the gene. ~9 Because none of the previous immunocytochemical studies distinguish among the -FMRFamide-conraining peptides encoded by the FMRFamide gene, the question of whether the FMRFamide polypeptide precursor undergoes post-translational processing on either a cellular or developmental level has not been addressed. To study the cellular expression pattern of a specific FMRFamide-containing peptide encoded by the Drosophila FMRFamide gene and address whether the FMRFamide gene undergoes differential post-translational processing, we have generated antisera specific to SDNFMRFamide. The antisera, purified on a peptide affinity column and characterized by preincubation with various

NEUROPEPTIDES peptides, were used to determine the spatial and temporal expression of SDNFMRFamide immunoreactive material. SDNFMRFamide immunoreactivity is first observed in the embryonic central nervous system in cells of the subesophageal ganglion and fibers that project from these cells into the brain and ventral ganglion. This same pattern of staining continues in neural tissue throughout development; however, no immunoreactivity is observed in gastrointestinal tissue. Double-label immunocytochemistry indicates that SDNFMRFamide immunoreactive material is present in cells stained by FMRFamide antisera. Based on position, SDNFMRFamide is expressed in a subset of cells previously identified as containing the FMRFamide polypeptide precursor. This result suggests that the Drosophila melanogaster FMRFamide polypeptide precursor undergoes differential posttranslational processing.

Materials and methods

Staging flies Drosophila rnelanogaster Oregon R flies were raised on cornmeal molasses media and maintained at 25°C. Central nervous systems were dissected from embryos collected on grape juice media plates at 13-16 and 16-20 hours. Larvae were collected by allowing flies to lay eggs on grape juice media plates for 21-22 h after which the flies were removed and all larvae were discarded. After 1 h, all larvae were collected; this was repeated over several hours to establish a range of ages. Tissue was dissected from 24, 48, and 72 h larvae. Prepupae were collected and central nervous systems dissected from 48, 72, and 94 hour old pupae. Adults were collected 4-6 h after eclosion and tissue was dissected from 1-2, 3-5, 5-7-day-old adults. All developmental stages were verified by morphological changes and no fewer than six preparations were examined for each time period.

Antisera production and purification The antigen, SDNFM-MAP, was synthesized with a Rainin Symphony multiple peptide synthesizer as a multiple antigenic peptide (MAP) which is composed of a core matrix of branching lysine onto

207

SPATIAL A N D TEMPORAL ANALYSIS OF S D N F M R F A M I D E

which the peptide was attached through the carboxyl terminus at eight branch sites. 2° The MAP was purified by preparative reversed-phase high performance liquid chromatography (HPLC) and the structure of the synthetic peptide was confirmed by mass spectrometry and amino acid analysis. Antisera were raised in two New Zealand white rabbits to the antigen with initial immunizations by subcutaneous injections at multiple sites of a total of 0.5 mg of antigen emulsified in Freund's complete adjuvant. Subsequent boosts were given every 2 weeks by subcutaneous injections of 0.25 mg of antigen in Freund's incomplete adjuvant. Antisera titers were analyzed by indirect immunofluorescence of whole mount third instar larval central nervous systems. 21 A peptide affinity column was made by coupling SDNFM-MAP to Affi-gel 10 (Bio-Rad Labs) in dry dimethyl sulfoxide and 1% triethylamine. The amount of peptide coupled to the affÉnityresin was 2 mg peptide/ml resin. To purify antisera, the resin was first washed with 10 column volumes of 5 mM sodium phosphate, pH 7.2, after which crude antisera diluted 1:1 with 10 mM sodium phosphate, pH 7.2, were applied to the column at a flow rate of 10 ml per h, and the resin was washed with 10 column volumes of 5 mM sodium phosphate, pH 7.2. SDNFM-antisera were eluted from the column with 5 column volumes of 0.1 M sodium citrate, pH 2.5, neutralized by collecting directly into 10 volumes of l M Tris, pH 8.0, and subsequently dialyzed against 41 of 0.005 mM sodium phosphate, pH 7.2, at 4°C for 24 h. The affinity-purified antisera were then aliquoted, freeze-dried, and stored at - 20°C.

Immunocytochemistry Tissue was dissected and prepared for immunocytochemistry according to previously published methods.~4'2~ Whole mount tissue preparations fixed in 4% paraformaldehyde or Bouin's (70% picric acid, 25% formalin, and 5% acetic acid) and washed in 0.5 M sodium phosphate, pH 7.2, containing 10% Triton X-100 and 1% sodium azide (PTN), were incubated with affinity-purified antisera, washed in PTN, and incubated with tetramethylinolocarbocyanine (CY3)-labeled donkey anti-rabbit antibody (Jackson Labs) or fluorescein

isothiocyanate (FITC)-labeled goat anti-rabbit antibody (Sigma). Double-label immunocytochemistryexperiments using SDNFM antisera and FMRFamide antisera (Peninsula Labs) were done as described above with the following modification: after incubation in the first primary antisera and fluorescently labeled antirabbit antibody, the tissue was incubated in preimmune rabbit antisera before incubation with the second primary antisera and fluorescently labeled anti-rabbit antibody.

Antisera specificity The specificity of the antisera was determined by the choice of antigen, affinity purification, and incubation of the antisera with peptides prior to immunocytochemistry. The antigen, SDNFMMAP, was designed to the N-terminal portion of SDNFMRFamide which avoids raising antisera to the C-terminal structure -RFamide common to numerous Drosophila neuropeptides. SDNFM antisera were purified from whole sera on a SDNFMMAP affinity column and control experiments included antisera preincubated with DPKQDFMRFamide, TPAEDFMRFamide, PDNFMRFamide, or SDNFM-MAP at 0.0001 M and 0.000001 M prior to use in immunocytochemical analysis.

Results

SDNFMRFamide immunoreactive material is observed in both cells and fibers that project from the cells throughout all developmental stages. The staining pattern is bilaterally symmetric to the midline and is consistent and reproducible for each developmental period. The cellular nomenclature for comparing the cells expressing SDNFMRFamide immunoreactivity to those expressing FMRFamide is from studies using FMRFamide antisera ~ 8 and antisera to the FMRFamide precursor. 19 SDNFMRFamide immunoreactivity is first observed in the embryonic central nervous system in multiple cells, termed SE cells, in the subesophageal ganglion and processes that extend from these cells into the brain and ventral ganglion (Fig.

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Fig. 1 SDNFMRFamide immunoreactivematerial in neural tissue dissectedfrom stage 16embryo.Cellsin the subesophageal ganglion (arrowheads) and fibers extendingfrom the cells into the brain and ventral ganglion are stained. The bar represents 50 microns. 1). The immunoreactive fibers that extend into the brain are positioned along the midline until the superior protocerebrum where they extend toward the central part of the brain lobe. The immunoreactive fibers that project into the ventral ganglion are positioned further from the midline and extend into the thoracic ganglia and abdominal ganglia. The staining of both the cells and fibers is strong. The p a t t e r n ' o f S D N F M R F a m i d e immunoreactivity observed in embryonic neural tissue is also seen in larvae - a cluster of SE cells in the subesophageal ganglion and fibers that project from these cells into the brain and ventral ganglion (Fig. 2). The immunoreactive fibers that extend from the cells into the brain and ventral ganglion in larval tissue are similar to those observed in embryonic

NEUROPEPT1DES tissue. Additional immunoreactive fibers extend to the ring gland in larval tissue dissected at 48 and 72 h, but not in tissue dissected at 24 h (Fig. 2). Although immunoreactive processes extend into the ventral ganglion and ring gland, no cell bodies are stained in these tissues. In addition, no immunoreactivity was observed in larval gastrointestinal tissue. In pupae (Fig. 3) and adult (Fig. 4), there is a similar pattern of cellular expression as seen in embryonic and larval tissue. However, there is a noticeable difference in the extensive arborization of the immunoreactive fibers present in the subesophageal ganglion and the thoracic ganglia of pupal and adult neural tissue. Although there are immunoreactive processes that extend into the pupal and adult ventral ganglion, no cells in the thoracic or abdominal ganglia were stained by S D N F M antisera. In addition, no immunoreactivity was observed in adult gastrointestinal tissue. Since our antigen included the N-terminus SDNFM-, but not the C-terminus -RFamide of S D N F M R F a m i d e , double-label immunocytochemical experiments were done to confirm that the cells containing S D N F M R F a m i d e immunoreactive material were also stained by antisera to FMRFamide. Neural tissue dissected from both larvae and adult were incubated with S D N F M antisera and F M R F a m i d e antisera and CY-3 and FITC-labeled second antibody, respectively. In both larval and adult neural tissue, SE cells in the subesophageal ganglion and processes projecting from these cells stained with S D N F M - antisera are also stained with F M R F a m i d e antisera (Fig. 5). Several MP cells in the medial protocerebrum and SP cells in the superior protocerebrum that have been identified as expressing D S K , 12'23'24 D M S , 21 and p r o F M R F a m i d e 19are stained by F M R F a m i d e antisera but not by S D N F M - antisera. In addition, those cells in the thoracic and abdominal ganglia that are stained by DSK, DMS, and F M R F a m i d e antisera, e.g. T1-3 and A8 cells, are not stained by S D N F M - anti sera. To characterize the specificity of the anfisera raised to S D N F M - M A P , antisera were preincubated with various peptides prior to use in immunocytochemistry. The peptides used were chosen from those encoded in the F M R F a m i d e polypeptide precursor. The pattern of

SPATIAL AND TEMPORAL ANALYSIS OF SDNFMRFAMIDE

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C Fig. 2 S D N F M R F a m i d e immunoreactive material in neural tissue dissected from larvae at 24, 48, and 72 h. Cells in the subesophageal ganglion (arrowheads) and fibers extending from the cells into the brain and ventral ganglion are stained. Additional fibers extend to the ring gland (arrows) at 48 and 72 h. The bar represents 50 microns.

210

Fig. 3 SDNFMRFamide immunoreactive material in neural tissue dissected from pupa at 72 h. Cells in the subesophageal ganglion (arrowheads) and fibers extending from the cells into the brain and ventral ganglion are stained. The bar represents 50 microns.

S D N F M R F a m i d e staining is not affected when the antisera are preincubated with D P K Q D F M R F amide or T P A E D F M R F a m i d e at 0.0001 M or 0.000001 M. A slight, overall reduction in staining intensity was observed when the antisera were preincubated with P D N F M R F a m i d e at 0.0001 M and 0.000001 M and all staining is abolished when the antisera is preincubated with the antigen, S D N F M MAP, at both 0.0001 M and 0.000001 M. N o difference in staining is observed when using antisera over a 500-fold concentration range or altering the immunocytochemical methodology, e.g. changing fixatives or detection methods.

Discussion

Numerous peptides have been identified in the central nervous system as messengers involved in important functions) 2 To learn more about the role(s) of peptidergic neurons in Drosophila brain we have undertaken the task to isolate and identify the naturally-occurring peptides containing -RF-

NEUROPEPTIDES

Fig. 4 SDNFMRFamide immunoreactive material in neural tissue dissected from adult at 3-5 days. Cells in the subesophageal ganglion (arrowheads) and fibers extending from the cells into the brain and ventral ganglion are stained. The bar represents 50 microns.

amide and to determine cellular expression using sequence-specific antisera. ~°'~'23'24 The expression pattern of specific neuropeptides used in conjunction with genetics and molecular studies may provide the opportunity to determine peptide function(s). Production of bioactive peptides from inactive polypeptide precursors involves a series of posttranslational modifications including proteolytic cleavage at basic residues and, frequently, the conversion of carboxy-terminal glycine-extended peptides to c~-amidated peptides. An organism can increase the diversity of its products by generating multiple peptides through post-translational proteolytic processing of a single polypeptide precursor. The release of these bioactive peptides can be regulated on a cell or developmental level via differential post-translational processing. The Drosophila melanogaster F M R F a m i d e

SPATIAL AND TEMPORAL ANALYSIS OF SDNFMRFAMIDE

211

nervous system and is similar throughout development - a cluster of cells in the subesophageal ganglion and fibers that project from these cells into the brain and ventral ganglion. This similarity in the pattern of expression throughout development suggests that SDNFMRFamide may act as an important transmitter or modulator. No immunoreactivity was observed in gastrointestinal tissue dissected from larva or adult. The characterization of the antisera specificity included the preincubation of the antisera with several different peptides chosen based on the structure of the FMRFamide polypeptide precursor. The inactivation of the antisera with the antigen but not DPKQDFMRFamide or TPAEDFMRFamide indicates that the affinity-purified antisera recognize SDNFMRFamide but not FMRFamide-containing peptides. While three peptides, SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide, encoded by Drosophila proFMRFamide have been isolated, a total of five different FMRFamidecontaining peptides can be predicted from the precursor. Because one of the predicted peptides, PDNFMRFamide, has a high degree of structure identity to SDNFMRFamide; we sought to determine whether antisera raised to SDNFM-MAP Fig. 5 Diagrams of larva (top) and adult (bottom) central might recognize PDNFMRFamide. Antisera were nervous system. A rectangle encloses that part of the nervous system represented in the double label immunocytochemistry preincubated with PDNFMRFamide or the anti(see Fig. 6). gen, SDNFM-MAP, at 0.0001 M and 0.000001 M and the staining patterns were compared. Preincubation with PDNFMRFamide and the antigen neuropeptide gene can be predicted to encode five did not have the same effect: while PDNFMRFdifferent FMRFamide-containing peptides some of amide only slightly reduced the overall staining, the which are present in multiple copies. Three of the antigen abolished all staining. This difference sugDrosophila FMRFamide gene products have been gests that the antisera has a higher avidity for isolated: SDNFMRFamide, DPKQDFMRF- SDNFMRFamide than for PDNFMRFamide but amide, and TPAEDFMRFamide. 11Two additional recognizes both peptides. Thus, ifPDNFMRFamide peptides, SPKQDFMRFamide and PDNFMRF- is processed from proFMRFamide, our results amide, can be predicted from proFMRFamide.9'rL identify cells which express SDNFMRFamide, To address whether the production of FMRF- PDNFMRFamide, or both SDNFMRFamide amide peptides is under cellular or developmental and PDNFMRFamide. The fact that SDNFMregulation, we have generated antisera using the antisera recognizes both SDNFMRFamide and antigen SDNFM-MAP and determined the devel- PDNFMRFamide does not change the interpretopmental expression pattern of SDNFMRFamide ation of the results of this study. Regardless of immunoreactive material in Drosophila melano- whether SDNFMRFamide and/or PDNFMRFyaster neural and gastrointestinal tissue. amide is present in an individual cell, the expression The expression of SDNFMRFamide immuno- pattern for SDNFMRFamide-like immunoreactive reactive material begins in the embryonic central material is a subset of cells previously reported to

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Fig. 6 SDNFMRFamide and FMRFamide double label immunocytochemistry in neural tissue from larva (top) and adult (bottom). SDNFMRFamide antisera are detected by CY3 (red) and FMRFamide antisera are detected by FITC (green). Those cells and fibers stained by both antisera are yellow. The rectangles on the schematics (Fig. 5) enclose the nervous system represented in the figures. Part of some cells stained only by FMRFamide antisera (green) in the superior protocerebrum appear yellow because of the overlap, at a different focal plane, of intensely staining SDNFMRFamide immunoreactive fibers (red). The bar represents 50 microns.

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SPATIAL AND TEMPORAL ANALYSIS OF SDNFMRFAMIDE contain proFMRFamide, FMRFamide

a result that suggests the

polypeptide precursor may undergo

differential post-translational processing.

11. 12.

Acknowledgements Peptides were synthesized and affinity resins were made at The University of Michigan Carbohydrate and Protein Structure Facility, Dr P. C. Andrews, Director. This work was supported by a NSF grant (IBN #9409623) to RN.

I3.

14.

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