ECB-ART-42412BMC Dev Biol 2012 Apr 27;12:14. doi: 10.1186/1471-213X-12-14.
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Histamine is a modulator of metamorphic competence in Strongylocentrotus purpuratus (Echinodermata: Echinoidea).
BACKGROUND: A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus. RESULTS: Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae. CONCLUSIONS: We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom--metamorphosis.
PubMed ID: 22541006
PMC ID: PMC3460732
Article link: BMC Dev Biol
Species referenced: Echinodermata
Genes referenced: hdc LOC100887844 LOC100893907 LOC115919910 LOC115925415 LOC762398
Antibodies: LOC503549 Ab1
Article Images: [+] show captions
|Figure 1. Pre-treatment of larvae with histamine (HA) leads to an increase of metamorphic competence based on a settlement induction assay with KCl (A) or settlement plates (SP) (B). Treatment of competent larvae with histamine (HA) receptor 1 antagonist (125 μM chlorpheniramine) and receptor 3 antagonist (125 μM thioperamide) leads to a reduction of arm length measured by the length of the exposed skeletal element from the extent of the arm flesh. HA receptor 2 antagonist (200 μM cimetidine) and HA (1 μM) did not significantly affect relative arm length (A). HA alone did not affect relative arm length in either experiment. Panel B illustrates the measurement of the arm flesh length subtracted from the skeletal element length, giving total exposed skeletal rod length. Note that larval arm length was measured within 24 hours of exposure to treatment. Panel C shows representative larvae that were measured in these experiments. Scale bar: 150 μm.|
|Figure 2. Pre-treatment of larvae with histamine (HA) leads to an increase of metamorphic competence based on a settlement induction assay with KCl (A) or settlement plates (SP) (B). Treatment of competent larvae with HA receptor 3 antagonist (C) and HA synthesis inhibitor (D) leads to induction of settlement. A cohort of larvae consisting of both, metamorphically competent and non-competent larvae (20% competence – (see Materials and Methods for details and replicates of experiment) were pre-treated with HA for 24 hours in filtered seawater in the complete absence of food and then exposed to the settlement induction assay. A) Larvae pre-exposed to HA settled at a significantly higher rate when exposed to KCl, a known artificial inducer of metamorphosis in sea urchin larvae compared to controls. (B) Larvae from the HA and control (no HA pre-exposure) did not undergo settlement without induction. After exposure to SP, histamine pre-treated larvae settled at a significantly higher rate compared to control larvae (no HA pre-exposure). We then tested the effect of histamine synthesis inhibitor and several HA receptor antagonists (note that larvae cohorts tested in these experiments were 80-100% competent). (C) Treatment of competent larvae with histamine receptor 3 antagonist (125 μM Thioperamide) for 24 hours leads to a significant increase in settlement without induction by KCl or SP. Histamine receptor 1 (125 μM chlorpheniramine) and receptor 2 (200 μM cimetidine) antagonists did not have any such direct effects on settlement rate. (D) Application of DL-alpha-methylhistidine (AMH, 100 μM), a known histidine decarboxylase (HDC) inhibitor to competent larvae for 72 h also led to a slight increase in settlement rate without induction.|
|Figure 3. Histamine (HA) distribution in embryonic stages of the sea urchinS. purpuratus. We used a polyclonal HA antibody for whole mount immunohistochemical (WMIHC) detection of HA in embryonic and post-embryonic (see Figure 4) stages. Panels A-F show late gastrula and early prism stages of the sea urchin S. pupuratus visualized using confocal microscopy. A-C are DIC (Differential Interference Contrast) images of panels D-F. Panels G-I are epiflourescent images of late prism to early pluteus stages. We detected two types of histaminergic cells indicated by white arrows in the panels. The first type are putative skeletal cells (SC) the second type are found at the base of the ciliated band (CBC). Note that we also found several unknown cell types (UC) showing HA immunoreactivity. In the early pluteus stages, cells at base of the ciliated band are clearly visible. We were also able to detect similar cells in the region of the aboral lip and in the ciliated epithelium of the larval arms POA – post oral arms). Early pluteus larvae also clearly show the development of the histaminergic lateral arm cluster (LAC). Blue represents a nuclear stain (DRAQ-5) and green indicates detection of HA antibody. Black arrows in DIC images (A-C) indicate corresponding regions of white arrows. Note that DIC images (A-C) are individual cross sections, while corresponding fluorescent images (D-F) are maximum projections. Therefore cells surface regions are not visible in all DIC images. The ubiquitous green stain on the right (BG) illustrates diffuse background fluorescence also seen in control samples (not shown). Scale bars: A,D - 30 μm; B,E - 35 μm; C,F - 25 μm; G,H,I - 20 μm, Abbreviations: S: Stomach; E: Esophagus; UC: Unkown cell types.|
|Figure 4. Histamine (HA) distribution in embryonic and post-embryonic stages of the sea urchinS. purpuratus. Panels A-F show larval stages with increasing age. Nonspecific staining is seen in the stomach (St) in panels A-D. A) 4 arm pluteus larva that shows the developing lateral arm cluster (LAC) at the base of the post-oral arms (POA; indicated by arrows). These cells project into the arms where histaminergic cells are visible in the region of the larval skeletons. HA is also detected in ciliated band cells (CBC) along the larval arms. B) At the 6 arm stage, the lateral cell clusters (LAC) have grown in size and extensive projections into both the anterior and posterior parts of the larva have developed. We also noticed an increase of histaminergic cells in the region of the larval skeletons and the CBC. Unknown cell types (UC) in the leftmost arm and the epaulette (Ep) also show some histaminergic immunoreactivity. Projections into the larval arms can be well seen in panels C and D) At metamorphic competence, a second cluster of histaminergic cells has developed in the region of the apical organ (AO). Extensive projections are now established in all parts of the larva. In addition, two large histaminergic cells were identified in the aboral arms (ALC). These cells are shown at a higher magnification in E. F) shows a different perspective of a competent larva, emphasizing the developing juvenile rudiment. Two main cell types appear to be histaminergic: the epineural folds (Ef) and the epidermis of the tube feet (Tf). The white arrow also depicts the histaminergic cells in the apical organ (AO). Scale bars:A - 75 μm; B - 90 μm; C - 90 μm; F - 150 μm; D - 150 μm; E - 50 μm.|
|Figure 5. The sea urchin histamine 1 receptor (suH1R) is localized in the plasma membrane of ectodermal cells in early pluteus stages. Whole mounted embryos were labeled with anti-suH1R antibodies (in red) and counterstained with Hoechst (in blue) to show nuclei. A and B show confocal images of the surface of the embryo; A’ and B’ show images taken at the equatorial plane; A” and B” show DIC images. St: Stomach; POA: Post-oral arm. Scale bars: 20 μm.|
|Figure 6. Histamine (HA) receptor 1 antagonist (125 μM chlorpheniramine) and histidine decarboxylase (HDC) inhibitor alpha-methylhistidine (AMH, 100 μM) treatment of competent larvae leads to increased caspase activity. Caspase activity was analyzed using FAM-VAD-FMK, a fluorescently tagged caspase inhibitor. Normalized fluorescence was measured in the arm tips of competent larvae. HA, HA receptor 2 antagonist (200 μM cimetidine), HA receptor 3 antagonist (125 μM Thioperamide) and KCl had no effect on caspase activity. The upper panel shows representative fluorescent images of treatment categories: B-control, C-HA (1 μM), D- HA receptor 1 antagonist (125 μM chlorpheniramine), E-HA receptor 2 antagonist (200 μM cimetidine), F-HA receptor 3 antagonist (125 μM Thioperamide), G-alpha-methylhistidine (AMH, 100 μM) and H-KCl. The lower panel shows the corresponding results of the fluorescent analysis. Panel A illustrates the approximate region of the arms that was included in the analysis. Note that all fluorescent intensities were normalized to the area measured and the exposure time. Scale bars: 20 μm.|
|Figure 7. Histamine receptor 1 antagonist (125 μM chlorpheniramine) did not affect caspase activity in pre-competent larvae. Caspase activity was analyzed using FAM-VAD-FMK, a fluorescently tagged caspase inhibitor. Overall fluorescence was measured in the arms tips of competent larvae. Histamine (1 μM), Histamine receptor 1 antagonist (125 μM chlorpheniramine) and histamine (1 μM) plus Histamine receptor 1 antagonist (125 μM chlorpheniramine) treatment had no significant effect on caspase activity. Scale bars: 20 μm.|
|Figure 8. Proposed model of modulatory actions in metamorphic competence and settlement of sea urchin larvae. Round symbols indicate agonistic relationships while flat lines indicate antagonistic interactions. Data presented here suggests a modulatory role of histamine (HA) in metamorphic competence. Based on our data we propose that HA leads to the acquisition of competence in pre-competent larvae and maintains competence in competent larvae by inhibiting caspase mediated apoptosis. Previously published data also suggest a function of thyroid hormones and nitric oxide (NO) signaling in modulating metamorphosis. Note that published data  established a link between NO and HA signaling in the sea urchin S. purpuratus. Future research will focus on the interactions of these signaling pathways in metamorphic competence and settlement|
References [+] :
Amarante-Mendes, Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death. 1999, Pubmed