Antibodies against conserved amidated neuropeptide epitopes enrich the comparative neurobiology toolbox
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Neuronal antibodies that show immunoreactivity across a broad range of species are important tools for comparative neuroanatomy. Nonetheless, the current antibody repertoire for non-model invertebrates is limited. Currently, only antibodies against the neuropeptide RFamide and the monoamine transmitter serotonin are extensively used. These antibodies label respective neuron-populations and their axons and dendrites in a large number of species across various animal phyla.
Several other neuropeptides also have a broad phyletic distribution among invertebrates, including DLamides, FVamides, FLamides, GWamides and RYamides. These neuropeptides show strong conservation of the two carboxy-terminal amino acids and are α-amidated at their C-termini. We generated and affinity-purified specific polyclonal antibodies against each of these conserved amidated dipeptide motifs. We thoroughly tested antibody reactivity and specificity both by peptide pre-incubation experiments and by showing a close correlation between the immunostaining signals and mRNA expression patterns of the respective precursor genes in the annelid Platynereis. We also demonstrated the usefulness of these antibodies by performing immunostainings on a broad range of invertebrate species, including cnidarians, annelids, molluscs, a bryozoan, and a crustacean. In all species, the antibodies label distinct neuronal populations and their axonal projections. In the ciliated larvae of cnidarians, annelids, molluscs and bryozoans, a subset of antibodies reveal peptidergic innervation of locomotor cilia.
We developed five specific cross-species-reactive antibodies recognizing conserved two-amino-acid amidated neuropeptide epitopes. These antibodies allow specific labelling of peptidergic neurons and their projections in a broad range of invertebrates. Our comparative survey across several marine phyla demonstrates a broad occurrence of peptidergic innervation of larval ciliary bands, suggesting a general role of these neuropeptides in the regulation of ciliary swimming.
Coronal ring nerve
Dorsal branch of the circum-oesophageal connectives
Differential interference contrast
Hours post fertilization
Red pigment concentrating hormone
Ventral branch of the circum-oesophageal connectives
Antibodies that show specific immunoreactivity across a broad range of species are valuable tools for comparative neuroanatomy in non-model organisms. For example, antibodies against serotonin commonly label cell bodies and their projections, allowing comparative studies of neurodevelopment and neuroanatomy across diverse species and phyla . Another commonly used antibody is that against FMRFamide, a neuropeptide first discovered in molluscs [2, 3]. Similar RFamide neuropeptides were later found to be widespread among eumetazoans [4–6]. A pioneering work reported the development of antibodies against the conserved amidated dipeptide motif RFamide . This RFamide and other FMRFamide antibodies have been extensively used in invertebrate neuroanatomy, owing to the broad distribution of RFamide-like peptides . The RFamide antibody labels distinct neuronal subsets and their projections, and can be applied as a neuronal marker to increase morphological resolution in complex adult tissues , or to reveal aspects of nervous system development and organization, allowing the clarification of phylogenetic relationships within phyla [10–12] or the study of nervous system evolution between related groups .
Neuropeptides are signalling molecules that are translated as precursor molecules, typically consisting of an N-terminal signal peptide and multiple copies of similar peptide motifs, flanked by dibasic cleavage sites (Lys and Arg residues). The precursor is cleaved and often further modified to yield shorter active neuropeptides [14, 15]. α-amidation is the most common post-translational modification, where a C-terminal glycine is enzymatically converted into an amide group. This modification protects the small peptides from degradation and is critical for receptor binding [16–18]. Amidation is also thought to confer high immunogenic potential to short neuropeptides [19–21] and antibodies raised against amidated peptides are highly specific for the amidated peptide moiety . Changes in hydrogen bonding capability caused by the amide group may lead to the improved receptor binding and increased immunogenicity of C-terminally amidated peptides .
The C-terminal residues in amidated neuropeptides are often highly conserved across different species and even phyla . We reasoned that, like the RFamide antibodies, other dipeptide antibodies could also potentially be used as neuronal markers across a wide range of species. Here we report the development of specific neuronal antibodies against the amidated dipeptide motifs of five conserved neuropeptides, DLamide, FVamide, FLamide, GWamide and RYamide. We show that these antibodies recognize specific subsets of neurons and their projections in cnidarian, annelid, mollusc, bryozoan and crustacean larvae. Furthermore, our antibody stainings reveal that the neuropeptidergic innervation of locomotor cilia is a general feature of ciliated larvae.
Generation of polyclonal neuropeptide antibodies
The amidated peptides, coupled to an adjuvant (lipoadjuvant Pam3) via an N-terminal cysteine (CRYamide, CGWamide, CFVamide, CFLamide, CDLamide), were used to immunize rabbits. Sera were affinity-purified on the respective peptide epitopes using a SulfoLink resin (Thermo Scientific, Rockford, USA) that allows the coupling of cysteine containing peptides via a disulphide bond. After coupling of 1 mg peptide epitope to 2 ml resin in Coupling Buffer (CB; 50 mM TRIS pH 8.5, 5 mM EDTA), the resin was washed three times with 10 ml CB. Excess reactive sites were blocked by incubating the resin in 2 ml 50 mM cysteine for 45 min, followed by three washes with 1 M NaCl and three washes with 25 ml phosphate buffered saline (PBS). Next, 25 ml serum was applied to the resin and this was incubated overnight to allow antibody binding. After flow-through of the serum, the resin was washed five times with 25 ml PBS followed by a wash with 15 ml 0.5 M NaCl/PBS and again twice with 10 ml PBS. The antibodies were eluted and fractionated with eight times 1 ml of 100 mM glycine pH 2.7, eight times 1 ml of 100 mM glycine pH 2.3 and eight times 1 ml of 100 mM glycine pH 2.0. The fractions were neutralized by directly collecting them in an adequate volume (about 40, 75 and 95 μl for the different pH solutions) of 1 M TRIS–HCl pH 9.5. The protein concentration of each fraction was determined, and the first two fractions of the pH 2.7 peak (usually fractions 2 and 3) were discarded, since these contained the lowest affinity antibodies. The peak fractions and the end-of-peak fractions were pooled, and concentrated, if necessary, using Vivaspin centrifugation tubes with a molecular weight cut-off of 10 kDa (Sartorius, Göttingen, Germany). Antibodies were stored in 50% glycerol at −20°C for mid-term (up to 1 year), and −80°C for long-term storage. A detailed protocol is available .
For immunostainings, larvae were fixed in 4% formaldehyde in PTW (PBS + 0.1% Tween-20) for 2 h and stored in 100% methanol at −20°C until use. After stepwise rehydration to PTW, samples were permeabilized with proteinase-K treatment (100 μg/ml in PTW, for 1 to 3 min). To stop proteinase-K activity, larvae were rinsed with glycine buffer (5 μg/ml in PTW) and post-fixed in 4% formaldehyde in PTW for 20 min followed by two 5 minwashes in PTW and two 5 minwashes in THT (0.1 M TRIS–HCl pH 8.5 + 0.1% Tween-20). Larvae and antibodies were blocked in 5% sheep serum in THT for 1 h. Primary antibodies were used at a final concentration of 1 μg/ml for rabbit neuropeptide antibodies and 0.5 μg/ml for mouse anti-acetylated tubulin antibody (Sigma, Saint Louis, USA) and incubated overnight at 6°C. Weakly bound primary antibodies were removed by two 10 min washes in 1 M NaCl in THT, followed by five 30 min washes in THT. Larvae were incubated overnight at 6°C in the dark in 1 μg/ml anti-rabbit Alexa Fluor® 647 antibody (Invitrogen, Carlsbad, CA, USA) and in 0.5 μg/ml anti-mouse FITC antibody (Jackson Immuno Research, West Grove, PA, USA) and then washed six times for 30 min with THT-buffer, and mounted in 87% glycerol including 2.5 mg/ml of the anti-photobleaching reagent 1,4-diazabicyclo[2.2.2]octane (Sigma, St. Louis, MO, USA). Pecten larvae were additionally treated with 4% paraformaldehyde in PBS with 50 μM EDTA pH 8.0 for 1 h to decalcify their shells before the immunostaining procedure (performed as described previously). For cnidarian larvae, we also used a mouse anti-tyrosylated tubulin antibody (Sigma, Saint Louis, USA) at 1 μg/ml. For immunostaining with multiple rabbit primary antibodies in the same sample, antibodies were directly labelled with a fluorophore using the Zenon® Tricolour Rabbit IgG Labelling Kit (Invitrogen, Carlsbad, CA, USA) and used in combination with mouse anti-acetylated tubulin antibody.
For blocking experiments, we pre-incubated the antibodies in 5 mM of the respective full-length Platynereis peptides (YYGFNNDLamide, AHRFVamide, AKYFLamide, VFRYamide, RGWamide) for 2 h before immunostainings.
Microscopy and image processing
Images were taken on an Olympus Fluoview-1000 confocal microscope (Olympus Deutschland GmbH, Hamburg, Germany) using a 60× water-immersion objective and the appropriate laser lines to capture fluorescent signals. Signals from RNA in situ hybridizations (nitro blue tetrazolium chloride/5-Bromo-4-cloro-3-indolyl phosphate precipitate) were imaged with reflection confocal microscopy as described . Images were processed with Imaris 6.4 (BitPlane Inc., Saint Paul, USA) and ImageJ 1.45 software . All image stacks are available .
Generation of specific antibodies against amidated dipeptide epitopes of neuropeptides
Rabbits were immunized with the short amidated peptides extended with an N-terminal cysteine to allow coupling to a carrier during the immunization procedure. We also used the cysteine residue to couple the peptides to a resin and to affinity purify the antibodies from the respective sera. We employed a high stringency affinity purification protocol including high salt washes and low pH elution to obtain high-affinity antibody fractions.
Overall, our specificity tests in Platynereis demonstrate that the antibodies raised against amidated dipeptide motifs are remarkably specific and can be used to obtain high-quality tissue stainings. To test the utility of our antibody collection as cross-species-reactive neuronal markers, we performed immunostainings on a variety of marine larvae from different species and phyla.
DLamide immunoreactivity in annelids
FVamide and FLamide immunoreactivity in annelids and molluscs
GWamide immunoreactivity in annelids, molluscs and crustaceans
RYamide immunoreactivity in cnidarian, annelid, bryozoan, mollusc and crustacean larvae
RYa neuropeptides have been described in a number of marine phyla, including cnidarians, annelids, molluscs, platyhelminthes and crustaceans. They are also present in terrestrial invertebrates, such as nematodes and insects (Figure 1E). In cnidarians, platyhelminthes and nematodes, RYa peptides co-occur with RFa peptides on the same precursor [33, 34, 41], whereas in most other phyla they originate from a distinct precursor. Given the broad phyletic distribution of RYa peptides and the observation that they often derive from distinct precursors expressed in different cells than RFa , the RYa antibody could have a great value for comparative neuroanatomical studies. To explore the potential of the RYa antibody, we tested its reactivity in cnidarians, annelids, molluscs, bryozoans and crustaceans.
Amidated dipeptide epitopes allow the generation of specific antibodies
Overall, our data argue that the antibodies strongly and specifically bind the amidated peptides we used for immunization. First, the stringent affinity purification protocol we employed together with the peptide-blocking experiments indicates that the antibodies strongly bind to the short amidated peptides. Second, the specific neuronal stainings in tissues corresponding to the expression patterns of the precursor genes in Platynereis show that the antibodies specifically bind to the respective peptides.
The strategy we employed to generate specific cross-species antibodies could also be applied to other conserved neuropeptides present in diverse taxa. With the increasing sampling of metazoan genomes and transcriptomes, and the accumulation of data from understudied groups (for example, hemichordates, platyhelminths, priapulids), we will have the chance to identify further conserved peptide motifs. Further sampling will also allow the identification of other taxonomic groups in which the antibodies described here could be used as neuronal markers. Given the brevity of the sequences, reactivity to multiple neuropeptide families with the same amidated termini cannot be excluded. For the proper interpretation of staining patterns, it is therefore also important to study mRNA expression and to scrutinize available transcriptomic and genomic resources. Importantly, our results show that these antibodies are not widely cross-reactive and do not recognize other amidated peptides. A single amino acid change seems to be sufficient to prevent antibody binding, since the DLa, FLa and FVa antibodies all recognize different cells.
Finally, C-terminal amidation is commonly used for immunization for peptides that derive from an internal part of the protein, to keep the peptide closer to its natural state. Our results caution that such an unnatural terminal amide in internal peptide sequences may trigger an undesired immune response, and potentially cause cross-reactivity to naturally occurring amidated peptides.
Cross-species antibodies suggest that the neuropeptidergic control of cilia is widespread in marine larvae
With the DLa, FVa and RYa antibodies, we commonly observe projections to larval ciliary bands. We have recently shown that the neurons expressing these neuropeptides also innervate the ciliary band in Platynereis larvae, and that these peptides regulate the activity of cilia. All three peptides increase the beating frequency of cilia and inhibit ciliary arrests, thereby influencing the swimming depth of planktonic Platynereis larvae . In Capitella larvae, all three neuropeptides are present in the ciliary band nerve. In Pecten, we found FVa and RYa immunoreactivity in nerves running along the ciliated velum, and in Phestilla in projections in the ciliated foot. RYa neurons also seem to innervate locomotor cilia in bryozoan and cnidarian larvae. This suggests that these peptides may also regulate ciliary activity in these larvae, indicating a general role for neuropeptides in the regulation of ciliary locomotion in marine invertebrate larvae .
We developed specific cross-species reactive antibodies that recognize the conserved neuropeptide motifs DLamide, FVamide, FLamide, GWamide and RYamide. These antibodies can be used in a wide range of marine invertebrates, including annelids, molluscs, bryozoans and cnidarians. Further genomic and transcriptomic sampling could identify other animal groups where these peptide motifs are conserved and where our antibodies could also be employed. Our work also highlights the antigenic potential of very short amidated peptide motifs. The ongoing sampling of neuropeptide diversity will allow the development of other similar antibodies, to enrich further the comparative neurobiology toolbox. Our sampling across diverse marine larvae demonstrates the broad utility of these antibodies, and also indicates that the neuropeptidergic regulation of ciliary locomotion may be a general feature of marine ciliated larvae.
We thank Harald Hausen for our Capitella stock and Andreas Bick for live Aurelia. We thank Elizabeth A. Williams for collecting Clava larvae and for comments on the manuscript, Nadine Randel and Michael G. Hadfield for Phestilla larvae, and Mechtild Seyboldt for collecting Pecten larvae. The Clava work was supported by an ASSEMBLE grant 227799. The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821.
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