Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae

The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well‐organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.     

Nervous system development of two crinoid species, the sea lily Metacrinus rotundus and the feather star Oxycomanthus japonicus

Nervous system development in echinoderms has been well documented, especially for sea urchins and starfish. However, that of crinoids, the most basal group of extant echinoderms, has been poorly studied due to difficulties in obtaining their larvae. In this paper, we report nervous system development from two species of crinoids, from hatching to late doliolaria larvae in the sea lily Metacrinus rotundus and from hatching to cystidean stages after settlement in the feather star Oxycomanthus japonicus. The two species showed a similar larval nervous system pattern with an extensive anterior larval ganglion. The ganglion was similar to that in sea urchins which is generally regarded as derived. In contrast with other echinoderm and hemichordate larvae, synaptotagmin antibody 1E11 failed to reveal ciliary band nerve tracts. Basiepithelial nerve cells formed a net-like structure in the M. rotundus doliolaria larvae. In O. japonicus, the larval ganglion was still present 1 day after settlement when the adult nervous system began to appear inside the crown. Stalk nerves originated from the crown and extended down the stalk, but had no connections with the remaining larval ganglion at the base of the stalk. The larval nervous system was not incorporated into the adult nervous system, and the larval ganglion later disappeared. The aboral nerve center, the dominant nervous system in adult crinoids, was formed at the early cystidean stage, considerably earlier than previously suggested. Through comparisons with nervous system development in other ambulacraria, we suggest the possible nervous system development pattern of the echinoderm ancestor and provide new implications on the evolutionary history of echinoderm life cycles.     

Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus

Deployment of the gene-regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an auto-repressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Fundamental aspects of arm repair phase in two echinoderm models

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored.Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools.Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models.Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.     

The biological regulation of sea urchin larval skeletogenesis – From genes to biomineralized tissue

Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.     

Cell lineage conversion in the sea urchin embryo

The mesoderm of the sea urchin embryo conventionally is divided into two populations of cells; the primary mesenchyme cells (PMCs), which produce the larval skeleton, and the secondary mesenchyme cells (SMCs), which differentiate into a variety of cell types but do not participate in skeletogenesis. In this study we examine the morphogenesis of embryos from which the PMCs have been removed microsurgically. We confirm the observation of Fukushi (1962) that embryos lacking PMCs form a complete skeleton, although in a delayed fashion. We demonstrate by microsurgical and cell marking experiments that the appearance of skeletogenic cells in such PMC-deficient embryos is due exclusively to the conversion of other cells to the PMC phenotype. Time-lapse video recordings of PMC-deficient embryos indicate that the converting cells are a subpopulation of late-ingressing SMCs. The conversion of these cells to the skeletogenic phenotype is accompanied by their de novo expression of cell surface determinants normally unique to PMCs, as shown by binding of wheat germ agglutinin and a PMC-specific monoclonal antibody. Cell transplantation and cell marking experiments have been carried out to determine the number of SMCs that convert when intermediate numbers of PMCs are present in the embryo. These experiments indicate that the number of converting SMCs is inversely proportional to the number of PMCs in the blastocoel. In addition, they show that PMCs and converted SMCs cooperate to produce a skeleton that is correct in both size and configuration. This regulatory system should shed light on the nature of cell-cell interactions that control cell differentiation and on the way in which evolutionary processes modify developmental programs.     

Vestigial ophiopluteal structures in the lecithotrophic larvae of Ophionereis schayeri (Ophiuroidea)

Evolution of echinoderm development from a feeding to a non-feeding mode can be examined by studying non-feeding larvae with structures that appear to be vestiges derived from a feeding ancestral state. The lecithotrophic larvae of the Australian brittle star Ophionereis schayeri possess such features, and the early development of this species was documented by light and scanning electron microscopy. The embryos undergo irregular cleavage, resulting in the formation of different sized blastomeres, with subsequent development through a wrinkled blastula stage. The lecithotrophic larva of O. schayeri possesses several vestigial ophiopluteal structures, including a continuous ciliated band, a larval gut, and a larval skeleton. The ciliated band is a reduced expression of the continuous ciliated band typical of ophioplutei. The larval gut is a transiently complete system, but an esophageal plug and rapid closure of the blastopore renders it nonfunctional. The larval skeleton, though reduced, consists of four rods corresponding to the body, posterolateral, anterolateral, and postoral rods characteristic of an ophiopluteus. Due to a heterochrony in larval skeletogenesis, the postoral rods develop early and simultaneously with the other rods. Compared with the larvae of other lecithotrophic ophiuroids, the larva of O. schayeri is one of the most reduced ophiopluteal forms reported to date.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

Arrays in rays: terminal addition in echinoderms and its correlation with gene expression

The echinoderms are deuterostomes that superimpose radial symmetry upon bilateral larval morphology. Consequently, they are not the first animals that come to mind when the concepts of segmentation and terminal addition are being discussed. However, it has long been recognized that echinoderms have serial elements along their radii formed in accordance with the ocular plate rule (OPR). The OPR is a special case of terminal growth, forming elements of the ambulacra that define the rays in echinoderms. New elements are added at the terminus of the ray, which may or may not be marked by a calcified element called the terminal plate (the "ocular" of sea urchins). The OPR operates in every echinoderm, from the occasionally bizarre fossils of the Cambrian to the most familiar extant taxa. Using the OPR and other criteria of recognition, echinoderm body wall can be divided into two main regions: extraxial components are associated with the somatocoels, axial components (formed in accordance with the OPR) with the hydrocoel. We compare patterns of development in axial regions of echinoderms with those found in the anterior-posterior axes of the earliest echinoderms as well as other invertebrates. Although axial and extraxial skeletons appear to be composed of the same biomineral matrix, the genes involved in patterning these two skeletal components are likely distinct. During development of the axial skeleton, for instance, the genes engrailed and orthodenticle are expressed in spatial and temporal patterns consistent with the OPR. Other genes such as distal-less seem to demarcate early ontogenetic boundaries between the axial rudiment and the extraxial larval body. There is a complex and pervasive reorganization of gene expression domains to produce the highly divergent morphologies seen in the Echinodermata. We integrate morphological and genetic information, particularly with respect to the origins of radial symmetry in the rudiment, and the concomitant development of the rays.     

Commensal vs. parasitic relationship between Carapini fish and their hosts: some further insight through δC and δN measurements

In the Moorea Lagoon (French Polynesia), the pearlfish Carapus boraborensis, Carapus homei, Carapus mourlani and Encheliophis gracilis are generally found inside echinoderm hosts such as the holothurian Bohadschia argus and the starfish Culcita novaeguineae. At the end of their larval stage, these fish settle on the reef and directly enter their echinoderm host where they undergo an important metamorphosis. The aim of this study was to get further insight on the type of symbiosis (commensal vs. parasite) between these fish and their hosts. δ¹⁵N and δ¹³C measurements were determined in the tissues of invertebrate hosts (holothurians and starfish) and carapids (larvae, juveniles and adults). The obtained isotopic signatures reveal different kinds of associations: metamorphosing larvae, juveniles and adults of C. boraborensis and C. homei do not feed at all on host holothurian tissues, C. mourlani and its asterian host display a commensal relationship without any feeding association, while E. gracilis is likely to feed on the tissue of the holothurian.     

SALMFamide neuropeptides cause relaxation and eversion of the cardiac stomach in starfish

Feeding in starfish of the species Asterias rubens involves eversion of the cardiac stomach over prey such as mussels and oysters. For eversion to be accomplished the cardiac stomach must be relaxed. Here we show that two neuropeptides (S1 and S2) belonging to a family of echinoderm neuropeptides called SALMFamides cause concentration-dependent relaxation of the cardiac stomach in vitro, with S2 being 10 to 20 times more potent than S1. Previously, we have obtained evidence that nitric oxide mediates neural control of cardiac stomach relaxation in Asterias. However, S2-induced relaxation of the cardiac stomach is not affected by an inhibitor of the nitric oxide ''receptor'' soluble guanylyl cyclase. Therefore, cardiac stomach relaxation in starfish appears to be controlled by at least two neural signalling pathways acting in parallel. To assess the involvement of the SALMFamides in mediating cardiac stomach eversion in Asterias, experiments were performed in which water (control) or S1 or S2 was injected into the perivisceral coelom. Cardiac stomach eversion was observed after 5 min in 3% of tests with water, in 11% of tests with S1 and in 57% of tests with S2. Importantly, the effectiveness of S1 and S2 in promoting eversion corresponds with their relative potency as cardiac stomach relaxants in vitro. Collectively, these data indicate that SALMFamide neuropeptides may be involved in regulating the process of cardiac stomach eversion in starfish.