Abstract
The nervous system of the free-living planktonic larvae of sea urchins is relatively “simple,” but sufficiently complex to enable sensing of the environment and control of swimming and feeding behaviors. At the pluteus stage of development, the nervous system comprises a central ganglion of serotonergic neurons located in the apical organ and sensory and motor neurons associated with the ciliary band and the gut. Neuropeptides are key mediators of neuronal signaling in nervous systems but currently little is known about neuropeptidergic systems in sea urchin larvae. Analysis of the genome sequence of the sea urchin Strongylocentrotus purpuratus has enabled the identification of 38 genes encoding neuropeptide precursors (NP) in this species. Here we characterize for the first time the expression of nine of these NP genes in S. purpuratus larvae, providing a basis for a functional understanding of the neurochemical organization of the larval nervous system. In order to accomplish this we used single and double in situ hybridization, coupled with immunohistochemistry, to investigate NP gene expression in comparison with known markers (e.g., the neurotransmitter serotonin). Several sub-populations of cells that express one or more NP genes were identified, which are located in the apica organ, at the base of the arms, around the mouth, in the ciliary band and in the mid- and fore-gut. Furthermore, high levels of cell proliferation were observed in neurogenic territories, consistent with an increase in the number of neuropeptidergic cells at late larval stages. This study has revealed that the sea urchin larval nervous system is far more complex at a neurochemical level than was previously known. Our NP gene expression map provides the basis for future work, aimed at understanding the role of diverse neuropeptides in control of various aspects of embryonic and larval behavior.
Highlights
The evolution of neuronal cell types and nervous systems is a hotly debated topic [1, 2], reflecting the diversity in nervous system organization found in animals
To obtain a detailed overview of neuropeptide precursors (NP) gene expression in the developing nervous system of the sea urchin embryo and larva, we first analyzed S. purpuratus genome sequence data to characterize the complexity of the neuropeptidome in this species
The latest version of the S. purpuratus genome (Echinobase, v 4.2; http://www. echinobase.org/Echinobase/) was analyzed using newly identified echinoderm NP sequences as queries for BlastP searches and SignalP (http://www.cbs.dtu.dk/services/SignalP-3.0/; [37]) was used to determine the presence of a signal peptide, which is a characteristic feature of neuropeptide precursors and other secreted proteins
Summary
The evolution of neuronal cell types and nervous systems is a hotly debated topic [1, 2], reflecting the diversity in nervous system organization found in animals. Expression of Sp-ILP1 in the gut is affected by different feeding regimes [26], highlighting an ancient deuterostome role of ILP secreted peptides and the power of echinoderms in helping resolve evolutionary questions. Against this background, there exists the opportunity to investigate the expression of multiple NP genes in populations of neurons in larval sea urchins, and to correlate findings with existing knowledge of the larval nervous system. The identification of specific populations of cells, neurons, and gut cells expressing NP genes enriches our understanding of the diversity of neuronal cell types in sea urchin larvae and the complexity of the larval nervous system
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