Figure 1.
The detection of melatonin by HPLC.
Overlaid chromatograms of a melatonin standard (green, 1×10−8 gr mL−1) and a sea anemone tissue extract (N. vectensis) with (black) and without (red) melatonin enrichment (1×10−8 gr mL−1). Chromatographic separation of tissue extracts was conducted based on fluorimetric detection (λex = 280 nm and λem = 345 nm). The mobile phase consisted of a mixture of 0.1% (v/v) formic acid in acetonitrile:water 17∶81 (v/v), which was delivered isocratically at a flow-rate of 1 mL min−1. The detailed conditions are described in the Materials and Methods.
Figure 2.
The detection of melatonin by HPLC/ESI/MS-MS.
The mass chromatogram that was obtained using the MRM detection mode (A), and positive-ion scan spectra of the melatonin parent (B) and daughter ions (C) in a representative sample of N. vectensis. Experimental conditions: C18 column; a mobile phase acetonitrile:water 17∶83 (v/v) that contained 0.1% formic acid and which was delivered isocratically; flow rate: 0.85 mL min−1; temperature: 30°C; injection volume: 10 µL; electrospray ionization in positive mode; mass spectrometric detection in multiple reaction monitoring mode (selected transitions: 233 to 216 and 233 to 174). The detailed conditions are described in the Materials and Methods.
Figure 3.
The distribution of melatonin immunoreactivity in Nematostella vectensis polyps.
(A) Overlaid fluorescent and differential interference contrast (DIC) micrographs of a young adult polyp that was stained with a melatonin-specific antibody (red). Melatonin accumulation is noticeable in the tentacle tips (tn), mesenteries (mes) and actinopharynx (ph). Oral ring (or). (B–C) Confocal micrographs of melatonin immunoreactivity in Nematostella sections. Melatonin appears to be unevenly distributed throughout the layers (arrow) of the hypostomal body wall (retracted polyp), implying layer-specific differences in melatonin sources/targets (B, C). (D) Increased magnification of extensive melatonin accumulation in the outer surfaces of the endothelium in the actinopharynx folds (arrowheads in B). (E) The punctate immunoreactive pattern lining the external surfaces of the gonads suggests a reproductive role for melatonin. (F) Uniform melatonin immunoreactivity was detected in gonadal endodermal cells. (G-I) Melatonin immunoreactivity was observed within both endodermal and ectodermal cells in the body wall; the endodermal signals were generally stronger. Occasionally, melatonin accumulated at the base of the endoderm, implying an interaction with epithelial muscular cells (arrowhead). Scale bars: B = 200 µm; A, E = 100 µm; C, D, F, G = 20 µm; I = 10 µm; H = 5 µm.
Figure 4.
Melatonin expression patterns in Nematostella vectensis developmental stages.
Confocal sections of whole mounts indicated that melatonin is enriched near the area of invagination in the early gastrula stage (A) and at the oral and aboral poles during the late gastrula stage of the forming planulae (B). In swimming planulae (older larvae with an actinopharynx and developing mesenteries), melatonin exhibited a definite early preference for the actinopharynx and the apical tuft (at), both of which are highly neuralized components of the developing nerve net (C). By the 4-tentacle primary polyp stage (D, E), this preference extends to include other neural areas, such as the tentacle (tn) tips (arrowheads) and mesenteries (mes, arrowhead). The concentration of melatonin in the developed mesenteries is very clear in (E). The asterisks denote the oral poles. Scale bars: A-E = 50 µm.
Figure 5.
The expression pattern of hydroxyindole-O-methyltransferase (HIOMT) mRNA in Nematostella vectensis.
Two representative HIOMT orthologs were evaluated using in situ hybridization (ISH) with specific probes. (A–C) The HIOMT expression pattern was similar between orthologs and indicated that melatonin is predominantly produced in the circumference of the actinopharynx (ph). High HIOMT expression levels were also evident in the endodermal layer of the hypostome (retracted individual, B). A higher magnification image of the outer surface of the endothelium in the actinopharynx folds (C). (D, E) Substantial HIOMT expression in reproductive tissues suggested that the considerable level of the melatonin that is observed in these tissues (see Fig. 1E–F) is locally produced. A uniform HIOMT expression pattern was observed among the cells. (F, G) In the body wall, predominantly endodermal HIOMT expression was observed throughout the apical end of the anemone and was uniformly distributed throughout the cells. This pattern differed from the pattern of melatonin immunoreactivity (see Fig. 1B–C). Note that melatonin production occurs also in tentacles (tn), albeit at lower levels. Endoderm (en), ectoderm (ec). Scale bars: A = 200 µm; B, C = 100 µm; E, F = 50 µm; D, G = 20 µm.
Figure 6.
Gene expression patterns of putative melatonin receptors in Nematostella vectensis.
Two representative melatonin receptor orthologs were evaluated using in situ hybridization (ISH) with specific probes. (A, B) A similar expression pattern between orthologs in adult polyps was observed and indicated that the mRNA for the putative receptor was predominantly expressed in the circumference of the actinopharynx (ph) and at substantially lower levels in the hypostome. (C) Higher magnification of the actinopharynx area indicated that the melatonin receptors are also expressed in the inner epithelium (arrowhead); albeit at lower levels (D) Substantial melatonin receptor expression was evident in fertile gonads (peripheral surfaces of endodermal cells), implying a reproduction-related receptor-mediated mechanism of action for melatonin. (E, F) The expression of putative melatonin receptors in the body wall was predominantly characterized by an endodermal distribution but was also sporadically observed at lower levels in both ectodermal cells and in the epithelial muscular layer (arrowheads) at the base of the endoderm. Scale bars: A = 500 µm; B = 200 µm; C = 100 µm; D, E = 50 µm; F = 20 µm.
Figure 7.
Confocal colocalization of melatonin and the Nematostella vectensis neural network in adult polyps.
(A) The distribution of melatonin immunoreactivity (red) generally corresponded with RFamide-expressing neurons (green). (B) Neuro-melatonin interactions were implied by specific colocalization (orange) along both the major longitudinal fasciculated neurite tracts and the minor inter-crossing neuronal pathways (arrowheads). (C) Melatonin distribution also paralleled the neural tracts in other areas of the N. vectensis neural net, such as the tentacles and the mesenterial endomesodermal cells that were proximate to the actinopharynx (arrowheads). (D, E) Confocal sections in the pharyngeal nerve ring area indicated neuro-melatonin associations. The asterisk denotes the oral pole. Scale bars: A = 500 µm; B, C-E = 200 µm.
Figure 8.
The colocalization of melatonin and the neural network in Nematostella vectensis sections.
(A–C) An overlay of FMRFamide (green) and melatonin (red), revealing little colocalization (orange) in the hypostome (A), extensive colocalization in the actinopharynx (ph) (B) and sporadic neuro-melatonin interactions in a subset of the fertile gonad cells (arrowheads) (C). (D–F) Pervasive colocalization of melatonin and FMRFamide was observed in several neurons in the body wall (arrowheads) (D); however, sensory cells (sc), ganglion cells (gc) and basiepithelial neurites (ne) were generally not melatonin-positive despite being located in highly melatonin-enriched surroundings (E–F). Ectoderm (ec). Scale bars: A, B = 50 µm; C, D = 20 µm; E, F = 10 µm.
Figure 9.
A schematic diagram of Nematostella illustrating the combinatorial expression of putative biosynthetic (HIOMT) and receptor gene elements overlaid with the distribution of melatonin immunoreactivity in different morphological features of the sea anemone.
The neural architecture in Nematostella (as assayed by FMRFamide, which is a known neuro-marker in the anthozoan nervous system [34]–[36]), overlaps with melatonin immunoreactivity in several areas. HIOMT expression patterns are highly correlated with melatonin immunoreactivity in key neural areas and reproductive tissues, corresponding to abundant expression of putative melatonin receptors. Varying levels in the amount/abundance of melatonin or mRNA expression of the gene elements among different body regions are schematically represented in the diagram by varying color intensities or by the number of consecutive cells that indicate the presence of a particular element.