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PLoS One
2013 Jan 01;89:e72797. doi: 10.1371/journal.pone.0072797.
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Nitric oxide acts as a positive regulator to induce metamorphosis of the ascidian Herdmania momus.
Ueda N
,
Degnan SM
.
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Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian metamorphosis.
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24019877
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Figure 1. A time course of Herdmania momus development indicating experimental strategies employed in this study.Developmental stages are indicated by hours post fertilisation (hpf) for embryonic and larval development. Post-larval development is indicated by hours post induction (hpi). All metamorphosis assays were initiated at competency (14 hpf). Grey shading indicates times at which RNA was sampled.
Figure 2. Effect of NOS inhibitors on metamorphosis of Herdmania momus.
(A) L-nitroarginine methyl ester (L-NAME), (B) aminoguanidine hemisulfate (AGH), and (C) S-methylisothiourea sulphate (SMIS) were applied at various concentrations to competent larvae (14 hpf). The number of individuals undergoing metamorphosis was counted 4 h after the initiation of exposure (4 hpi). Filtered sea water (FSW) and 40 mM KCl-elevated FSW were used as negative and positive controls, respectively. Data are presented as the mean percentage of larval metamorphosis ± SEM (n = 3 for each treatment, 30 larvae per replicate). Diamonds indicate the actual percentages of larval metamorphosis in each replicate. Letters above error bars indicate statistically significant differences (P<0.05), as determined by one-way analysis of variance and Tukey’s HSD post hoc testing.
Figure 3. Effect of NO donors on metamorphosis of Herdmania momus.
(A) S-nitroso-N-acetyl-penicillamine (SNAP), (B) N-hydroxy-nor-arginine (nor-NOHA), and (C) L-Arginine were applied at various concentrations to competent larvae (14 hpf). The number of individuals undergoing metamorphosis was counted 4 h after the initiation of exposure (4 hpi). FSW and 40 mM KCl-elevated FSW were used as negative and positive controls, respectively. Data are presented as the mean percentage of larval metamorphosis ± SEM (n = 3 for each treatment, 30 larvae per replicate). Diamonds indicate the actual percentages of larval metamorphosis in each replicate. Letters above error bars indicate statistically significant differences (P<0.05), as determined by one-way analysis of variance and Tukey’s HSD post hoc testing.
Figure 4. Effect of heat-shock on metamorphosis of Herdmania momus.
Larvae were exposed at competency (14 hpf) to one of four temperatures (25, 29, 32 and 35°C) for 2 h. The number of individuals undergoing metamorphosis was counted 24 h after the initiation of heat-shock exposure (24 hpi). FSW and 40 mM KCl-elevated FSW, maintained at the culture temperature of 25°C, were used as negative and positive controls, respectively. Data are presented as the mean percentage of larval metamorphosis ± SEM (n = 3 for each treatment, 30 larvae per replicate). Diamonds indicate the actual percentages of larval metamorphosis in each replicate. Letters above error bars indicate statistically significant differences (P<0.05), as determined by one-way analysis of variance and Tukey’s HSD post hoc testing.
Figure 5. Normal (25°C) and abnormal (32°C) post-larval development of Herdmania momus.
(A) Normal 4 hpi post-larva. Black eyespots and ampullae growth are visible at the anterior end, left. (B) Normal 24 hpi post-larva. Ampullae are fully extended, bottom. (C) Normal 84 hpi post-larva. Adult morphology has been formed by this time; branchial basket and siphons can clearly be seen. (D) Abnormal 4 hpi post-larva. Arrow indicates incompletely resorbed tail at posterior end. (E) Abnormal 24 hpi post-larva. Arrow indicates some larval tail still remaining. (F) Abnormal 84 hpi post-larva. Arrow indicates some larval tail still remaining; arrowhead indicates deformed branchial basket. Scale bars: A-F, 100 µm.
Figure 6.
HmNOS and HmHSP90 gene expression through Herdmania momus normal development.Transcript abundance was assessed by qRT-PCR using mRNA purified from a pool of ∼200 embryos or larvae for each developmental stage (red circles). Transcript abundance in pooled samples of spontaneously metamorphosed post-larvae is denoted by blue diamonds. Data are presented as log-transformed mean ± SEM of three technical replicates.
Figure 7. Effect of NO-disrupting pharmacological agents on HmNOS and HmHSP90 gene expression.Competent larvae (14 hpf) were exposed to either 10 mM L-NAME (NOS inhibitor) or 0.1 mM SNAP (NO donor), or were not exposed (untreated control), and sampled 4 hours later as either larvae (18 hpf; had not initiated metamorphosis) or post-larvae (4 hpi; had initiated metamorphosis). All samples depicted here are thus of the same developmental age. Transcript abundance was assessed by qRT-PCR using mRNA purified from a pool of larvae (red circles) or post-larvae (blue diamonds). Data are presented as log-transformed mean ± SEM of three technical replicates.
Figure 8. Effect of heat-shock on HmNOS and HmHSP90 gene expression.Competent larvae (14 hpf) were exposed to one of four heat-shock temperatures (29, 32 and 35°C) or retained at the control temperature (25°C) for 2 h. Samples for RNA were collected at the start of the experiment as larvae (14 hpf), at 2 h after the initiation of the heat shock (2 hpi) as either larvae (had not initiated metamorphosis) or post-larvae (had initiated metamorphosis), and at 24 h after the initiation of the heat shock (24 hpi) as either larvae (had not initiated metamorphosis) or post-larvae (had initiated metamorphosis). Transcript abundance was assessed by qRT-PCR using mRNA purified from pools of larvae or post-larvae. Data are presented as log-transformed mean ± SEM of three technical replicates.
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