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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.
Figure 1. Quantitative PCR (QPCR) reveals that NP gene expression correlates with an increase in the complexity of the larval nervous system. NP gene expression (transcripts per embryo) is shown from the appearance of the first neuronal precursor cells at mesenchyme blastula stage [24 h post-fertilization (hpf)], through embryonic development to the early larval stage (70 hpf) when several types of neurons are differentiated. An increase in expression is seen for most NP genes from 48 to 70 hpf, when the first serotonergic neurons differentiate. The line graphs depict different levels of gene expression for 27 NP genes, with lower expression in graphs (A,C) and higher expression in graphs (B,D). The color of the NP gene name above each graph reflects the line color in the corresponding graph.
Figure 2. Expression of NP genes in the apical plate and post-oral neuron-like precursor cells of the gastrula embryo. Merged and single channel confocal images of 48 hpf embryos labeled using double fluorescent ISH are shown. (A) Expression of serotonin and Sp-FSALMFa highlights two populations present at 48 hpf, the serotonergic (white arrowheads) and post-oral (yellow arrowheads) neuron-like precursor cells, respectively. (B,C) Co-expression of three NP genes in the apical plate sensory precursors. (D,E) Co-expression of three NP genes in two-three post-oral neuron-like precursor cells. Bottom-left/Top-right corner indicates the probe or antibody used. Dotted white boxes highlight the magnified region shown to the right. Scale bars: 20 μm.
Figure 3. Expression of NP genes in the apical plate serotonergic neurons and ciliary band neuron-like cells. Merged and single channel confocal images of pluteus larvae labeled using double fluorescent ISH and immunohistochemistry are shown. (A) EdU-and SynB (1E11) labeling experiment showing a high rate of cell proliferation in the neurogenic territories (apical plate and ciliary band, lip and foregut). (B,C) Labeling of the larval nervous system using antibodies to serotonin and to acetylated tubulin (AcTub) and using Sp-SecV ISH. (B–G) NP gene expression in the apical plate serotonergic neurons. (H–J)
Sp-Trh and Sp-FSALMFa co-expressed in the ciliary band, at the base of the oral distal arms, adjacent to the apical plate. Bottom-left corner indicates the probe or antibody used. Top-right corner indicates the larval stage in hours' post-fertilization; bottom-left corner indicates the probe or antibody used. Dotted white boxes highlight the magnified region shown to the right. Scale bars: 20 μm.
Figure 4. Expression of NP genes in two clusters of two-three lateral ganglia neuron-like cells. Merged confocal images of pluteus larvae labeled using single and double FISH are shown. (A)
Sp-SecV and Sp-AN NP gene expression in lateral ganglia neuron-like cells. (B) Magnification of Sp-AN NP gene expression in the lateral ganglia neuron-like cells. Bottom-left corner indicates the probe used. Top-right corner indicates the larval stage in hours' post-fertilization. Dotted white boxes highlight the magnified region shown to the right. Scale bars: 20 μm.
Figure 5. Expression of NP genes in the mouth and gut cell systems. (A–F,I) Merged and single channel confocal images of pluteus larvae labeled using single and double fluorescent ISH. (G,H) Chromogenic whole-mount ISH of Sp-Np20 and Sp-Kp NP gene expression in pluteus larvae. Bottom-left corner indicates the probe used. Top-right corner indicates the larval stage in hours or weeks post-fertilization. Dotted white boxes highlight the magnified region shown to the right. Asterisk indicates the midgut. Scale bars: 20 μm.
Figure 6. Color coded cartoon map showing various combinations of NP gene and Sp-SecV expression at single cell resolution in the sea urchin at the gastrula stage and in pluteus larvae. (A) NP gene expression in two populations of cells at the gastrula stage. Serotonergic neuronal precursor cells (blue) and post-oral neuronal-like precursor cells in the oral ectoderm (magenta). (B,C) NP gene expression in eight different neuronal and presumed neuronal populations in the larval nervous system, shown from abanal and an oral view. Sensory serotonergic neurons (blue), lateral ganglion neurons (purple), presumed oral distal neurons (orange), presumed post-oral neurons (yellow), presumed lip neurons (red and pink), and mid-gut exocrine-like cells (dark green). Black and gray neurons (in foregut, ciliary band, and serotonergic interneurons) have been identified in previous studies but do not express any of the NP genes examined in this study. Abbreviated labels refers to the following, AP (Apical Plate), AbE (Aboral Ectoderm), OE (Oral Ectoderm), A (Archenteron), AO (Apical Organ), CB (Ciliary band), M (Mouth), FG (Foregut), MG (Midgut) and HG (Hindgut).
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