Cholinergic regulation of the sea urchin embryonic and larval development

Choline esters of polyenoic fatty acids block cleavage divisions of sea urchins and evoke the formation of one-cell multinuclear embryos. If the fatty acids AA-Ch or DHA-Ch are added at the mid or late blastula stage, many cells are extruded, forming extra-embryonic cell clusters near the animal pole of embryos or larvae. Both effects are prevented by dimethylaminoethyl esters of polyenoic fatty acids (AA-DMAE or DHA-DMAE) or their 5-hydroxytryptamides. Nicotinic acetylcholine receptor antagonists, imechine, d-tubocurarine or QX-222 provide partial protection against AA-Ch or DHA-Ch. The organophosphate pesticide, chlorpyrifos, or a combination of (-)-nicotine + phorbol 12-myristate 13-acetate, also evoke the mass extrusion of transformed embryonic cells at the animal pole of larvae. These effects are similarly antagonized by AA-DMAE, DHA-DMAE, or fatty acids 5-hydroxytryptamides. Taking together, these results suggest that AA-Ch and DHA-Ch act on sea urchin embryos and larvae as agonists of acetylcholine receptors, whereas AA-DMAE and DHA-DMAE act as antagonists. The ability of fatty acids 5-hydroxytryptamides to prevent the effects of AA-Ch or DHA-Ch may be due to restoration of the normal dynamic balance of cholinergic and serotonergic signaling during cleavage divisions and gastrulation.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Par6 regulates skeletogenesis and gut differentiation in sea urchin larvae

Partitioning-defective (par) genes were originally identified as genes that are essential for the asymmetric division of the Caenorhabditis elegans zygote. Studies have since revealed that the gene products are part of an evolutionarily conserved PAR-atypical protein kinase C system involved in cell polarity in various biological contexts. In this study, we analyzed the function of par6 during sea urchin morphogenesis by morpholino-mediated knockdown and by manipulation swapping of the primary mesenchyme cells (PMCs). Loss of Par6 resulted in defects in skeletogenesis and gut differentiation in larvae. Phenotypic analyses of chimeras constructed by PMC swapping showed that Par6 in non-PMCs is required for differentiation of archenteron into functional gut. In contrast, Par6 in both PMCs and ectodermal cells cooperatively regulates skeletogenesis. We suggest that Par6 in PMCs plays an immediate role in the deposition of biomineral in the syncytial cable, whereas Par6 in ectoderm may stabilize skeletal rods via an unknown signal(s).     

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn’t affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cells of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.     

The evolution of sea urchin sperm bindin

Sea urchins have been model organisms for the study of fertilization for more than a century. Fertilization in sea urchins happens externally, which facilitates the study of sperm-egg attachment and fusion, and means that all of the molecules involved in gamete recognition and fusion are associated with the gametes. Sea urchin sperm bindin was the first "gamete recognition protein" to be isolated and characterized (Vacquier and Moy 1977), and bindin has since been studied by developmental biologists interested in fertilization, by biochemists interested in membrane fusion and by evolutionary biologists interested in reproductive isolation and speciation. Research on bindin was last reviewed thirteen years ago by Vacquier et al. (1995) in an article titled "What have we learned about sea urchin sperm bindin?" in which the authors reviewed the identification, isolation and early molecular examinations of bindin. Research since then has focused on bindin's potential role in fusing egg and sperm membranes, comparisons of bindin between distantly related species, studies within genera linking bindin evolution to reproductive isolation, and studies within species looking at fertilization effects of individual bindin alleles. In addition, the egg receptor for bindin has been cloned and sequenced. I review this recent research here.     

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.     

Morphological evolution in sea urchin development: hybrids provide insights into the pace of evolution

Hybridisations between related species with divergent ontogenies can provide insights into the bases for evolutionary change in development. One example of such hybridisations involves sea urchin species that exhibit either standard larval (pluteal) stages or those that develop directly from embryo to adult without an intervening feeding larval stage. In such crosses, pluteal features were found to be restored in fertilisations of the eggs of some direct developing sea urchins (Heliocidaris erythrogramma) with the sperm of closely (Heliocidaris tuberculata) and distantly (Pseudoboletia maculata) related species with feeding larvae. Such results can be argued to support the punctuated equilibrium model—conservation in pluteal regulatory systems and a comparatively rapid switch to direct development in evolution. 1 , 1 Generation of hybrids between distantly related direct developers may, however, indicate evolutionary convergence. The ‘rescue’ of pluteal features by paternal genomes may require maternal factors from H. erythrogramma because the larva of this species has pluteal features. In contrast, pluteal features were not restored in hybridisations with the eggs of Holopneustes purpurescens, which lacks pluteal features. How much of pluteal development can be lost before it cannot be rescued in such crosses? The answer awaits hybridisations among indirect and direct developing sea urchins differing in developmental phenotype, in parallel with investigations of the genetic programs involved. BioEssays 26:343–347, 2004. © 2004 Wiley Periodicals, Inc.     

Microheterogeneity of late histones in larval stages of sea urchin development

• 1.1. The total histone complement of early plutei were compared with that of intermediate and late larvae of the sea urchin Tetrapygus niger.
• 2.2. Electrophoretic comparison indicates that there are quantitative and qualitative shifts of the five classes throughout late larval development.
• 3.3. The strong similarity in the amino acid composition of total histones isolated from early, intermediate and late plutei indicates that the observed electrophoretic heterogeneity is due to post-translational modifications.

     

Micromere-derived signal regulates larval left-right polarity during sea urchin development

The micromeres (Mics) lineage functions as a morphogenetic signaling center in early embryos of sea urchins. The Mics lineage releases signals that regulate the specification of cell fates along the animal-vegetal and oral-aboral axes. We tested whether the Mics lineage might also be responsible for differentiation of the left-right (LR) axis by observing of the placement of the adult rudiment, which normally forms only on the left side of the larvae, after removal of the Mics lineage. When all of the Mics lineage were removed from embryos of the regular sea urchin Hemicentrotus pulcherrimus between the 16- and 64-cell stages, the LR placement of the rudiment became randomized. However, the immediate retransplantation of the Mics rescued the normal LR placement of the rudiment, indicating that the Mics lineage releases a signal that specifies LR polarity. Additionally, we investigated whether the specification of LR polarity of whole embryos in the indirect-developing sea urchin H. pulcherrimus is affected by LiCl exposure, which disturbs the establishment of LR asymmetry in a direct-developing sea urchin. Larvae derived from normal animal caps combined with LiCl-exposed Mics descendants were defective in normal LR placement of the rudiment, suggesting that LiCl interferes with the Mics-derived signal. In contrast, embryos of two sand dollar species (Scaphechinus mirabilis and Astriclypeus manni) were resistant to alteration of LR placement of the rudiment by either removal of the Mics lineage or LiCl exposure. These results indicate that the Mics lineage is involved in specification of LR polarity in the regular sea urchin H. pulcherrimus, and suggest that LiCl impairs the normal LR patterning by affecting Mics-derived signaling.     

Novel origins of lineage founder cells in the direct-developing sea urchin Heliocidaris erythrogramma

The lineage and fate of each blastomere in the 32-cell embryo of the direct-developing sea urchin Heliocidaris erythrogramma have been traced by microinjection of tetramethylrhodamine-dextran. The results reveal substantive evolutionary modifications of the ancestral cell lineage pattern of indirect sea urchin development. Significant among these modifications are changes in the time and order of cell lineage segregation: vegetal ectodermal founder cells consistently arise earlier than during indirect development, while internal founder cells generally segregate later and in a different sequence. Modifications have also arisen in proportions of the embryo fated to become various cell types and larval structures. Ectodermal fates, particularly vestibular ectoderm, comprise a greater proportion of the total cellular volume in H. erythrogramma. Among internal cell types, coelom consumes more and endoderm less of the remaining cellular volume than during indirect sea urchin development. Evolutionary modifications are also apparent in the positional origin of larval cell types and structures in H. erythrogramma. These include an apparent tilt in the axis of prospective cell fate relative to the animal-vegetal axis as defined by cleavage planes. Together these evolutionary changes in the cell lineage of H. erythrogramma produce an accelerated loss of dorsoventral symmetry in cell fate relative to indirect development. The extent and diversity of rearrangements in its cell lineage indicate that the non-feeding larva of H. erythrogramma is a highly modified, novel form rather than a degenerate pluteus larva. These same modifications underscore the evolutionarily flexible relationship between cell lineage, gene expression, and larval morphology in sea urchin development.     

The effect of salinity on larval survival and development in the sea urchin Echinometra lucunter

Summary

Reductions in salinity can have adverse effects on larval development and larval survival in some invertebrate taxa but not others. Salinity tolerance of larvae may be particularly important in echinoderms because they are both poor ion regulators and stenohaline. I examined the effect of six levels of salinity (15, 18, 21, 24, 27 and 33 PSU) on survival and rate of development of larvae in the subtropical sea urchin Echinometra lucunter. In the short-term, mortality rate was significantly lower in 33 PSU than in all other salinities except 27 PSU, and it was significantly greater in 15 and 18 PSU than in all higher salinities. In the long-term, daily and cumulative mortality were significantly greater in 15 PSU than in most other salinities over 11 days of development (except for cumulative mortality in 18 PSU). They were significantly greater in 18 PSU than in 21 PSU or 33 PSU over a period of 13 days. Furthermore, daily mortality was significantly greater in 18 PSU than in 24 PSU or 27 PSU at 13 d after fertilization. Daily and cumulative mortality were significantly lower in 33 PSU than in 21, 24 or 27 PSU over a period of 17 days. Although in the control (33 PSU) 75% of larvae completed development to the 8-arm stage at 35 d, no larvae developed further than the 4-arm stage in 18, 21, 24 or 27 PSU; in 15 PSU, ~60% of larvae did not develop further than swimming blastulae. Since prolonged exposure to salinities as high as 27 PSU (frequently recorded in the adult habitat) can result in great larval losses, adaptive behaviours that prevent larvae from entering water layers of low salinity will enhance their chance for survival.     

Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs

Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.     

A large calcium-binding protein associated with the larval spicules of the sea urchin embryo

A tissue-specific, high molecular weight, calcium-binding protein from the sea urchin embryo is described. This protein, designated as CBP 180, has a molecular weight of 180,000 under reducing conditions, and is extractable with 1% Triton X-100. It accumulates rapidly during development, starting roughly at the onset of spiculogenesis. When embryos are cultured in the presence of inhibitors of spicule formation, such as tunicamycin and zinc ions, accumulation of CBP180 is depressed or stopped. By immunofluorescence technique and by using an antibody specifically generated against this protein, CBP180 is mainly localized in primary mesenchyme cells and spicular syncytium of the pluteus larva. Little or none is detectable in ectoderm, endoderm or blastocoelar extracellular matrix. These results suggest that the protein is involved in calcium sequestration in the differentiation of larval spicules.     

Relative importance of parental and larval nutrition on larval development and metamorphosis of the sea urchin Strongylocentrotus droebachiensis

We examined the relative importance of parental nutritional condition and larval food ration on the rates of development, growth and metamorphosis of larvae of Strongylocentrotus droebachiensis (Müller) in a laboratory experiment. Parents were reared for 22 months on either a high ration of kelp (Laminaria spp., 6 days week⁻¹) supplemented with mussel flesh (Mytilus spp., 1 day week⁻¹) (KM), or a low ration of kelp (1 day week⁻¹) (KL). Larvae were fed either a high ration (5000 cells ml⁻¹) or a low ration (500 cells ml⁻¹) of microalgae (Dunaliella tertiolecta). Larval food ration had a strong effect on the rates of development, growth, and metamorphosis, which were all significantly greater in larvae fed the high ration. Test diameter of settlers also was significantly greater in the high than the low ration. Parental nutritional condition had little or no effect on the rates of development and growth, and no effect on settler size. The rate of metamorphosis was significantly higher in larvae from the KM than the KL treatment in the high but not the low ration (where rates of metamorphosis were similar). Although parental condition generally had a small effect on larval development, our results suggest that when planktonic food is abundant, larvae of adults from nutritionally rich habitats (such as kelp beds) may metamorphose sooner than those of adults from nutritionally poor habitats (such as barrens).