Antibodies against conserved amidated neuropeptide epitopes enrich the comparative neurobiology toolbox

Abstract

BackgroundNeuronal 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.ResultsSeveral 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.ConclusionsWe 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.

Global view of the evolution and diversity of metazoan neuropeptide signaling

“…Wamides may have been ancestrally involved in mediating life-cycle transitions triggered by chemosensory cues (73). RFamides may have ancient roles in muscle control (74), ciliary locomotion (27,62), and food intake (75). The role of glycoprotein hormones and insulin-like peptides in cnidarians is unclear, but they may be part of a small-peptide/glycoprotein/insulin module in the regulation of growth, metabolism, or sexual maturation.…”

“…pNPs are often repetitive; the number and length of repeats change during evolution, or the sequences diverge into distinct peptides within a precursor (21)(22)(23). Conserved sequence stretches in pNPs often constitute only a few residues corresponding to biologically active short peptides (24)(25)(26)(27). Neuropeptides showing such limited conservation were nevertheless shown to be ligands of orthologous GPCRs in different phyla, confirming that the pNPs are orthologous (28)(29)(30).…”

Conserved MIP receptor–ligand pair regulates Platynereis larval settlement

“…Rabbit antisera against Platynereis MIP7 and against the conserved C-terminal VWamide motif were affinity-purified by using the respective peptides coupled to a SulfoLink resin (Thermo Scientific) via an N-terminal Cys (CAWNKNSMRVWamide and CVWamide) as previously described (60). In situ hybridization was performed as previously described (39).…”

A “Love” Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails

“…Other peptides within the precursor protein include the buccalin peptides, which have been implicated in depressing muscle contractions in Aplysia and as neuromodulators given their widespread distribution in nervous tissue (34 -36). This function is supported by the presence of the C-terminal GLamide, which is a feature also found in insect allotostatin A (37) and in the class of DLamides identified in the annelid Platyneiris demurilii (38). The buccalin gene of T. pisana has widespread expression outside the mucous gland, including the CNS of aestivated (dormant in warm conditions) snails, where the buccalin-2 isoform is distributed within regions that control several physiological roles (33).…”