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.
Highlights
Neuronal antibodies that show immunoreactivity across a broad range of species are important tools for comparative neuroanatomy
We have recently shown in the marine annelid model Platynereisdumerilii that the precursor mRNAs for these neuropeptides are expressed in largely non-overlapping subsets of neurons in the larval episphere
None of these neuropeptides co-expresses with FMRFamide in Platynereis [23], suggesting that antibodies against their conserved amidated dipeptides could substantially increase the number of neurons that can be labelled in other species
Summary
Neuronal antibodies that show immunoreactivity across a broad range of species are important tools for comparative neuroanatomy. Antibodies against serotonin commonly label cell bodies and their projections, allowing comparative studies of neurodevelopment and neuroanatomy across diverse species and phyla [1]. Another commonly used antibody is that against FMRFamide, a neuropeptide first discovered in molluscs [2,3]. A pioneering work reported the development of antibodies against the conserved amidated dipeptide motif RFamide [7] This RFamide and other FMRFamide antibodies have been extensively used in invertebrate neuroanatomy, owing to the broad distribution of RFamide-like peptides [8]. 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 [9], or to reveal aspects of nervous system development and organization, allowing the clarification of phylogenetic relationships within phyla [10,11,12] or the study of nervous system evolution between related groups [13]
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