Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
???displayArticle.abstract???
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.
???displayArticle.pubmedLink???
24019877
???displayArticle.pmcLink???PMC3760835 ???displayArticle.link???PLoS One
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 (nâ=â3 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 Tukeyâs 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 (nâ=â3 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 Tukeyâs 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 (nâ=â3 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 Tukeyâs 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 â¼200 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.
Akool,
Nitric oxide increases the decay of matrix metalloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR.
2003, Pubmed
Akool,
Nitric oxide increases the decay of matrix metalloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR.
2003,
Pubmed
Amador-Cano,
Role of protein kinase C, G-protein coupled receptors, and calcium flux during metamorphosis of the sea urchin Strongylocentrotus purpuratus.
2006,
Pubmed
,
Echinobase
Arnold,
Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae.
1997,
Pubmed
Baxter,
G protein and diacylglycerol regulate metamorphosis of planktonic molluscan larvae.
1987,
Pubmed
Bender,
Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo.
1999,
Pubmed
Biggers,
Settlement and Metamorphosis of Capitella Larvae Induced by Juvenile Hormone-Active Compounds Is Mediated by Protein Kinase C and Ion Channels.
1999,
Pubmed
Bishop,
Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).
2008,
Pubmed
Bishop,
On nitric oxide signaling, metamorphosis, and the evolution of biphasic life cycles.
2003,
Pubmed
,
Echinobase
Bishop,
Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.
2001,
Pubmed
Bishop,
NO/cGMP signaling and HSP90 activity represses metamorphosis in the sea urchin Lytechinus pictus.
2001,
Pubmed
,
Echinobase
Boyle,
An inexpensive alternative to glassmilk for DNA purification.
1995,
Pubmed
Brown,
Hsp90--from signal transduction to cell transformation.
2007,
Pubmed
Brüne,
Nitric oxide: NO apoptosis or turning it ON?
2003,
Pubmed
Cáceres,
Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75.
2011,
Pubmed
Chambon,
Tail regression in Ciona intestinalis (Prochordate) involves a Caspase-dependent apoptosis event associated with ERK activation.
2002,
Pubmed
Chambon,
ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles.
2007,
Pubmed
Chung,
Nitric oxide as a bioregulator of apoptosis.
2001,
Pubmed
Colasanti,
Nitric oxide in invertebrates.
1998,
Pubmed
Colasanti,
Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.
1995,
Pubmed
Comes,
Regulatory roles of nitric oxide during larval development and metamorphosis in Ciona intestinalis.
2007,
Pubmed
Degnan,
Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae.
1999,
Pubmed
Degnan,
Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus.
1997,
Pubmed
Dehal,
The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.
2002,
Pubmed
Ercolesi,
Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis.
2012,
Pubmed
Eri,
Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis.
1999,
Pubmed
Filippov,
Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells.
1997,
Pubmed
Froggett,
Metamorphosis in the Marine Snail Ilyanassa obsoleta, Yes or NO?
1999,
Pubmed
García-Cardeña,
Dynamic activation of endothelial nitric oxide synthase by Hsp90.
1998,
Pubmed
Grumbach,
A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription.
2005,
Pubmed
Gupta,
Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species.
1995,
Pubmed
Hadfield,
Why and how marine-invertebrate larvae metamorphose so fast.
2000,
Pubmed
He,
Nitric oxide represses the Arabidopsis floral transition.
2004,
Pubmed
Kashiwagi,
Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis.
1999,
Pubmed
Kawashima,
Dynamic changes in developmental gene expression in the basal chordate Ciona intestinalis.
2005,
Pubmed
Kim,
The regulatory role of nitric oxide in apoptosis.
2001,
Pubmed
Kim,
Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling.
1999,
Pubmed
Kim,
Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms.
1997,
Pubmed
Kroiher,
Heat shock as inducer of metamorphosis in marine invertebrates.
1992,
Pubmed
Leise,
An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system.
2000,
Pubmed
Li,
Nitric oxide as a modulator of apoptosis.
2005,
Pubmed
Lindquist,
The heat-shock response.
1986,
Pubmed
Nakayama,
Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis.
2001,
Pubmed
Nakayama,
Further characterization of genes expressed during Ciona intestinalis metamorphosis.
2002,
Pubmed
Nakayama-Ishimura,
Delineating metamorphic pathways in the ascidian Ciona intestinalis.
2009,
Pubmed
Palumbo,
Nitric oxide in marine invertebrates: a comparative perspective.
2005,
Pubmed
Park,
Nitric oxide limits transcriptional induction of nitric oxide synthase in CNS glial cells.
1994,
Pubmed
Pechenik,
Nitric oxide inhibits metamorphosis in larvae of Crepidula fornicata, the slippershell snail.
2007,
Pubmed
Pfaffl,
Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.
2002,
Pubmed
Queitsch,
Hsp90 as a capacitor of phenotypic variation.
2002,
Pubmed
Rutherford,
Hsp90 as a capacitor for morphological evolution.
1998,
Pubmed
Schmidt,
Physiologic and proteomic evidence for a role of nitric oxide in biofilm formation by Nitrosomonas europaea and other ammonia oxidizers.
2004,
Pubmed
Serfözö,
Nitric oxide level regulates the embryonic development of the pond snail Lymnaea stagnalis: pharmacological, behavioral, and ultrastructural studies.
2002,
Pubmed
Song,
Heat-shock protein 90 augments neuronal nitric oxide synthase activity by enhancing Ca2+/calmodulin binding.
2001,
Pubmed
Stuehr,
Enzymes of the L-arginine to nitric oxide pathway.
2004,
Pubmed
Tao,
Nitric oxide, an endogenous regulator of Dictyostelium discoideum differentiation.
1997,
Pubmed
Taris,
Inhibitory function of nitric oxide on the onset of metamorphosis in competent larvae of Crepidula fornicata: A transcriptional perspective.
2009,
Pubmed
Taylor,
Nitric oxide down-regulates hepatocyte-inducible nitric oxide synthase gene expression.
1997,
Pubmed
Tenu,
Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages.
1999,
Pubmed
Tomanek,
Evolutionary and acclimation-induced variation in the heat-shock responses of congeneric marine snails (genus Tegula) from different thermal habitats: implications for limits of thermotolerance and biogeography.
1999,
Pubmed
Vandesompele,
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.
2002,
Pubmed
Wilken,
Suppression of mycelia formation by NO produced endogenously in Candida tropicalis.
1999,
Pubmed
Woods,
Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein.
2004,
Pubmed
Yeyati,
Hsp90 selectively modulates phenotype in vertebrate development.
2007,
Pubmed
Yoshida,
Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase.
2003,
Pubmed
Young,
Hsp90: a specialized but essential protein-folding tool.
2001,
Pubmed
Zhang,
The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite.
2012,
Pubmed
