Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

A laboratory study of the correlation between texture and the speed of locomotion by the sea urchin Hemicentrotus pulcherrimus

This laboratory study examined the relationship between substrate texture and movement speed of the sea urchin Hemicentrotus pulcherrimus. We assessed the movement speed of 14 sea urchins placed on either acrylic, or three types of waterproof sandpaper, on the bottom of a water tank. Images were taken at regular intervals and analyzed to determine the speed of each sea urchin. Light intensity was stronger at one end of the tank. Our analysis showed sea urchins moved away from light, at a speed that was negatively correlated with the roughness of the substrate, with slower movement on rougher surfaces. This result has implications for the design of equipment for capturing sea urchins in areas where their explosive population growth presents a threat to algal growth and reef environments.     

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor‐β (TGF‐β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock‐in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet‐1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate‐based screening and identified BMP type I receptor kinase inhibitors LDN‐193189 and DMH1 as activators of luciferase. LDN‐193189 induced ES cells to express the genes encoding Pet‐1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF‐β receptor inhibitor SB‐431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN‐193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF‐β target gene Lefty, whereas the opposite effect was observed with SB‐431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF‐β receptor signaling are implicated in serotonergic differentiation.

     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

Can multitrophic interactions and ocean warming influence large‐scale kelp recovery?

Ongoing changes along the northeastern Atlantic coastline provide an opportunity to explore the influence of climate change and multitrophic interactions on the recovery of kelp. Here, vast areas of sea urchin‐dominated barren grounds have shifted back to kelp forests, in parallel with changes in sea temperature and predator abundances. We have compiled data from studies covering more than 1,500‐km coastline in northern Norway. The dataset has been used to identify regional patterns in kelp recovery and sea urchin recruitment, and to relate these to abiotic and biotic factors, including structurally complex substrates functioning as refuge for sea urchins. The study area covers a latitudinal gradient of temperature and different levels of predator pressure from the edible crab (Cancer pagurus) and the red king crab (Paralithodes camtschaticus). The population development of these two sea urchin predators and a possible predator on crabs, the coastal cod (Gadus morhua), were analyzed. In the southernmost and warmest region, kelp forests recovery and sea urchin recruitment are mainly low, although sea urchins might also be locally abundant. Further north, sea urchin barrens still dominate, and juvenile sea urchin densities are high. In the northernmost and cold region, kelp forests are recovering, despite high recruitment and densities of sea urchins. Here, sea urchins were found only in refuge habitats, whereas kelp recovery occurred mainly on open bedrock. The ocean warming, the increase in the abundance of edible crab in the south, and the increase in invasive red king crab in the north may explain the observed changes in kelp recovery and sea urchin distribution. The expansion of both crab species coincided with a population decline in the top‐predator coastal cod. The role of key species (sea urchins, kelp, cod, and crabs) and processes involved in structuring the community are hypothesized in a conceptual model, and the knowledge behind the suggested links and interactions is explored.     

Ion flow regulates left–right asymmetry in sea urchin development

The degree of conservation among phyla of early mechanisms that pattern the left–right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H⁺/K⁺-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H⁺/K⁺-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H⁺ and/or K⁺, and also of Ca²⁺, functions in the establishment of LR asymmetry in sea urchin development.Electronic Supplementary Material Supplementary material is available for this article at     

Predator‐induced selection on urchin activity level depends on urchin body size

Temporally consistent individual differences in behavior impact many ecological processes. We simultaneously examined the effects of individual variation in prey activity level, covering behavior, and body size on prey survival with predators using an urchin–lobster system. Specifically, we tested the hypothesis that slow‐moving purple sea urchins (Strongylocentrotus purpuratus) and urchins who deploy extensive substrate (pebbles and stones) covering behavior will out‐survive active urchins that deploy little to no covering behavior when pitted against a predator, the California spiny lobster (Panulirus interruptus). We evaluated this hypothesis by first confirming whether individual urchins exhibit temporally consistent differences in activity level and covering behavior, which they did. Next, we placed groups of four urchins in mesocosms with single lobster and monitored urchin survival for 108 hr. High activity level was negatively associated with survival, whereas urchin size and covering behavior independently did not influence survival. The negative effect of urchin activity level on urchin survival was strong for smaller urchins and weaker for large urchins. Taken together, these results suggest that purple urchin activity level and size jointly determine their susceptibility to predation by lobsters. This is potentially of great interest, because predation by recovering lobster populations could alter the stability of kelp forests by culling specific phenotypes, like foraging phenotypes, from urchin populations.     

Consumers of sea urchins,<Emphasis Type="Italic"> Paracentrotus lividus</Emphasis> and<Emphasis Type="Italic"> Arbacia lixula</Emphasis>, in shallow Mediterranean rocky reefs

Underwater observations on fish and asteroid consumers (i.e. predators and scavengers) of sea urchins, Paracentrotus lividus and Arbacia lixula, were carried out at several locations in shallow Mediterranean rocky reefs. Observations conducted in the marine reserve of Torre Guaceto (Adriatic Sea) revealed that sparid fishes, Diplodus sargus and D. vulgaris, are the main fish predators of small (<1 cm in test diameter) and medium (1–4 cm) sea urchins, whereas the labrids Coris julis and Thalassoma pavo preyed only upon small sea urchins. Large D. sargus were able to prey upon small and medium, and occasionally large (>4 cm) sea urchins, whereas medium and small Diplodus preyed mainly upon small sea urchins. The number of sea urchins preyed upon by fishes was negatively related to sea urchin size for both species. P. lividus appeared to be subject to higher predation levels than A. lixula. The scavenger guild comprised 11 fish species, with D. sargus, D. vulgaris, Coris julis and Chromis chromis accounting for about 80% of scavenger fishes. Observations performed at several locations in the Mediterranean on the predatory asteroid Marthasterias glacialis revealed that only 3% of the detected individuals were preying upon sea urchins. Due to the importance of sea urchins for assemblage structure and functioning of Mediterranean rocky reef ecosystems, these results may have also important implications for management of fishing activities.Communicated by H.-D. Franke     

The efficiency and effectiveness of different sea urchin removal methods for kelp forest restoration

Sea urchin overgrazing has caused widespread phase shifts from kelp forests to “urchin barrens” on many temperate reefs, reducing habitat complexity, productivity, and biodiversity. Sea urchin removal is increasingly used for kelp restoration; however, few studies have quantified the efficiency and effectiveness of different removal methods, resulting in limited understanding of their practicality. In this study, the efficiency (removal rate) and effectiveness (proportion removed) of four removal methods were evaluated in northeastern New Zealand. We compared culling or collecting sea urchins by either SCUBA or freediving in 128 small-scale plots (25 m²). We also evaluated the efficiency and effectiveness of culling in four large (1.6–2 ha) barren areas, scales relevant for restoration. On average, culling sea urchins was 1.9–4.4 times faster than collecting, and SCUBA was 1.5–3.3 times faster than freediving. Removal rates increased with sea urchin density, especially for culling on SCUBA, while freediving removal rates increased with experience. Effectiveness was lower in large-scale removals (86–93% of sea urchins ≥40 mm removed) compared to small-scale removals (98–99%), but sufficient for restoration objectives. Estimated time per area (using SCUBA culling) was similar across large-scale removals (49–57 hours/ha), despite an almost 2-fold variation in initial sea urchin densities (approximately 4–8 urchins/m²), suggesting area may better predict total removal time than simply number of sea urchins across low-density ranges. While sea urchin removal provides a rapid, feasible, and effective approach to restoring kelp in urchin barrens, restoration plans need to also address the causes of sea urchin overpopulation to ensure long-term benefits.     

Isolation and characterization of fucose sulfate from jelly coat glycoprotein of sea urchin egg

Fucose sulfate was isolated from the egg jelly glycoproteins of two kinds of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, by mild acid hydrolysis and paper chromatography followed by charcoal and Sephadex G-25 column chromatography. The yields of fucose sulfate were 24 and 20% of the total fucan sulfate from H. pulcherrimus and P. depressus, respectively.On the basis of chemical analysis, periodate oxidation and infrared spectroscopy, the structure of the fucose sulfate of the jelly coat glycoproteins derived from two kinds of sea urchins was proposed to be L-fucose-4-sulfate.     

dbEST-derived SSR markers in sea urchin (Hemicentrotus pulcherrimus)

Sea urchin is an important model organism for evolutionary biology, embryology, and gene regulation study. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus. Characteristics of nine EST-SSR loci were investigated using 41 Hemicentrotus pulcherrimus individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.122 to 0.7805, while the expected heterozygosity ranged from 0.4472 to 0.7696. These loci and markers will be useful for population genetics and systemic evolution among species of sea urchin.     

Development of nervous systems to metamorphosis in feeding and non-feeding echinoid larvae,the transition from bilateral to radial symmetry

The development of nervous system (NS) in the non-feeding vestibula larva of the sea urchin, Holopneustes purpurescens, and the feeding echinopluteus larva of Hemicentrotus pulcherrimus was examined by focusing on fate during metamorphosis. In H. purpurescens, the serotonergic NS (SerNS) appeared simultaneously and independently in larval tissue and adult rudiment, respectively, from 3-day post-fertilization. In 4-day vestibulae, an expansive aboral ganglion (450 × 100 μm) was present in the larval mid region that extended axons toward the oral ectoderm. These axons diverged near the base of the primary podia. An axonal bundle connected with the primary podia and the rim of vestopore on the oral side. Thus, the SerNS of the larva innervated the rudiment at early stage of development of the primary podia. This innervation was short-lived, and immediately before metamorphosis, it disappeared from the larval and adult tissue domains, whereas non-SerNS marked by synaptotagmin remained. The NS of 1-month post-fertilization plutei of H. pulcherrimus comprised an apical ganglion (50 × 17 μm) and axons that extended to the ciliary bands and the adult rudiment (AR). A major basal nerve of serotonergic and non-serotonergic axons and a minor non-serotonergic nerve comprised the ciliary band nerve. In 3-month plutei, axonal connection among the primary podia in the neural folds completed. The SerNS never developed in the AR. Thus, there was distinctive difference between feeding- and non-feeding larvae of the above sea urchins with respect to SerNS and the AR.     

Alginate degradation by bacteria isolated from the gut of sea urchins and abalones

Degradation of alginate and its constituents, polymannuronate (polyM) and polyguluronate (polyG), by gut bacteria isolated from sea urchins and abalones in the northern part of Japan, were investigated. Bacterial counts in the guts of sea urchin S. intermedius, were 10⁵ to 10⁸ CFU/g, and in abalone H. discus hannai, counts ranged from 10⁶ to 10⁹ CFU/g. More than 80% of total 600 isolates were found to have alginolytic activity. The alginolytic bacteria were predominantly fermentative, but some differences were observed in their substrate specificity as well as between the flora in the gut of sea urchins and the abalones. Seventy percent of the alginolytic bacteria from the sea urchins showed no degrading preference for polyM or polyG blocks, and were able to degrade both the substrates simultaneously. Most of the alginolytic bacteria (96.6%) from sea urchins belonged to the genus Vibrio. The majority of alginolytic bacteria (68.0% on average) from abalones only degraded polyG and they were predominantly non-motile fermenters. From these results, it appeared that a different type of association exists between alginolytic gut microflora and the marine algal feeders with respect to the level of contribution by bacteria to the host's digestion of alginate. Correspondence to: T. Sawabe     

Increased macroalgal abundance following mass mortalities of sea urchins (Strongylocentrotus droebachiensis) along the Atlantic coast of Nova Scotia

Summary Recurrent outbreaks of disease between 1980 and 1983 caused catastrophic mortality of sea urchins (>260,000 t fresh weight) along 280 km (straight line distance) of the Atlantic coast of Nova Scotia. The complete elimination of sea urchins and concomitant development of fleshy macroalgal communities have occurred along different parts of this coast in different years. Macroalgal communities in areas where sea urchins died off 1, 3 and 4 years previously are compared to existing sea urchin-dominated barren grounds and to a mature kelp bed without sea urchins. Changes in macroalgal cover and species composition, and increases in biomass, density and size of kelp (Laminaria) species, characterize the succession from barren grounds to 3- and 4-year-old kelp beds. The greatest change occurred between one and three years following sea urchin mass mortality. Within 3 years, kelp beds attained a level of biomass (7.6 kg m^-2) comparable to that of mature beds. Recovery of sea urchin populations via recruitment of planktonic larvae has been slow and spatially variable. Large-scale reciprocal fluctuations in kelp and sea urchin biomass may characterize the trajectory of a dynamic system which cycles between two alternate community states: kelp beds and sea urchin-dominated barren grounds. Periodic decimation of sea urchin populations by disease may be an important mechanism underlying this cyclicity.     

Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex

Rho‐associated kinase (ROCK) plays a critical role in pressure overload‐induced left ventricular remodelling. However, the underlying mechanism remains unclear. Here, we reported that TGF‐β1‐induced ROCK elevation suppressed BMP‐2 level and strengthened fibrotic response. Exogenous BMP‐2 supply effectively attenuated TGF‐β1 signalling pathway through Smad6‐Smurf‐1 complex activation. In vitro cultured cardiomyocytes, mechanical stretch up‐regulated cardiac TGF‐β1, TGF‐β1‐dependent ROCK and down‐regulated BMP‐2, but BMP‐2 level could be reversed through blocking TGF‐β1 receptor by SB‐431542 or inhibition of ROCK by Y‐27632. TGF‐β1 could also activate ROCK and suppress endogenous BMP‐2 level in a dose‐dependent manner. Knock‐down BMP‐2 enhanced TGF‐β1‐mediated PKC‐δ and Smad3 signalling cascades. In contrast, treatment with Y‐27632 or SB‐431542, respectively suppressed ROCK‐dependent PKC‐δ and Smad3 activation, but BMP‐2 was only up‐regulated by Y‐27632. In addition, BMP‐2 silencing abolished the effect of Y‐27632, but not SB‐431542 on suppression of TGF‐β1 pathway. Further experiments showed that Smad6 Smurf1 interaction were required for BMP‐2‐evoked antagonizing effects. Smad6 overexpression attenuated TGF‐β1‐induced activation of PKC‐δ and Smad3, promoted TGF‐β RI degradation in BMP‐2 knock‐down cardiomyocytes, and could be abolished after knocking‐down Smurf‐1, in which Smad6/Smurf1 complex formation was critically involved. In vivo data showed that pressure overload‐induced collagen deposition was attenuated, cardiac function was improved and TGF‐β1‐dependent activation of PKC‐δ and Smad3 was reduced after 2 weeks treatment with rhBMP‐2(0.5 mg/kg) or Y‐27632 (10 mg/kg) in mice that underwent surgical transverse aortic constriction. In conclusion, we propose that BMP‐2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload‐induced cardiac fibrosis.     

Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.     

Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC₅₀ (EC₅₀ = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.     

A halogenated amino acid-containing sperm activating peptide and its related peptides isolated from the egg jelly of sea urchins,Tripneustes gratilla,Pseudoboletia maculata,Strongylocentrotus nudus,Echinometra mathaei and Heterocentrotus mammillatus

• 1.1. Twenty-eight peptides were isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Strongylocentrotus nudus, Echinometra mathaei (type A and B) and Heterocentrotus mammillatus and their amino acid sequences were determined.
• 2.2. Two of the peptides obtained from T. gratilla egg jelly possessed a bromophenylalanine (Br-Phe) residue in their sequences (Gly-(Br-Phe)-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly and Gly-(Br-Phe)-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly).
• 3.3. All of the peptides elevated cyclic GMP concentrations in the spermatozoa of the respective sea urchin and caused a shift in the apparent mol. wt of a major sperm protein of the respective sea urchin.
• 4.4. They stimulated respiration rates of the spermatozoa of Hemicentrotus pulcherrimus as well as their own species.
• 5.5. One-half maximal concentrations of the peptides for respiratory stimulation of H. pulcherrimus spermatozoa were between 10⁻¹¹ M and 10⁻⁹ M except a methionine-containing peptide which was about 10⁻⁷ M.