The Sea Urchin Blastula: Extent of Cellular Determination

The extent of cellular determination at the blastula stage ofsea urchins has been investigated by using as parameters themorphological differentiation and cellular interactions of dissociatedblastula cells under conditions that either promote or discouragetheir reaggregation and reconstitution into embryos. The dissociatedblastula cells have the capacity to differentiate into at leastthree different specific cell types that occur in the normallydeveloping sea urchin embryo. Of these, ciliated epitheliumis known to originate in the animal half, and spindle-shapedcells and polyfilamentous cells are of mesenchymal origin andare derived from the vegetal half. The formation of ciliatedepithelium and blastulation requires calcium and hyalin; thecellular differentiation and interactions of the mesenchymalcells do not. This basic difference in calcium requirement isexplored in terms of differential hyalin synthesis or secretionby the cells at either equatorial half of the embryo. The animalizingeffect of zinc and the vegetalizing effect of lithium can beexplained in part by their enhancement and reduction, respectively,of hyalin gelation by calcium. Literature relating to otherbasic differences along the animal-vegetal axis of the embryois appraised. These differences include the use and the assemblyof microtubule protein and quantitative RNA synthesis duringboth the cleavage and blastula stages.     

Characterization of microsatellite loci for the Australian sea urchin Heliocidaris erythrogramma

We report 16 polymorphic microsatellite loci from Heliocidaris erythrogramma, a common sea urchin endemic to temperate Australian waters. These microsatellites were tested in a minimum of 30 individuals, which yielded between five and 14 alleles per locus. Expected heterozygosity ranged from 0.52 to 0.92 with four loci deviating from Hardy-Weinberg expectations. These markers are expected to be useful for experimental studies involving paternity analysis and for quantifying population structure in H. erythrogramma across its geographic range.     

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.     

Maternal factors and the evolution of developmental mode: Evolution of oogenesis in Heliocidaris erythrogramma

 Evolutionary change in developmental mode in sea urchins is closely tied to an increase in maternal provisioning. We examined the oogenic modifications involved in production of a large egg by comparison of oogenesis in congeneric sea urchins with markedly different sized oocytes and divergent modes of development. Heliocidaris tuberculata has small eggs (95 μm diameter) and the ancestral mode of development through feeding larvae, whereas H. erythrogramma has large eggs (430 μm diameter) and highly modified non-feeding lecithotrophic larvae. Production of a large egg in H. erythrogramma involved both conserved and divergent mechanisms. The pattern and level of vitellogenin gene expression is similar in the two species. Vitellogenin processing is also similar with the gonads of both species incorporating yolk protein from coelomic and hemal stores into nutritive cells with subsequent transfer of this protein into yolk granules in the developing vitellogenic oocyte. Immunocytology of the eggs of both Heliocidaris species indicates they incorporate similar levels of yolk protein. However, H. erythrogramma has evolved a highly divergent second phase of oogenesis characterised by massive deposition of non-vitellogenic material including additional maternal protein and lipid. Maternal provisioning in H. erythrogramma exhibits recapitulation of the ancestral vitellogenic program followed by a novel oogenic phase with hypertrophy of the lipogenic program being a major contributor to the increase in egg size. Received: 12 August 1998 / Accepted: 25 November 1998     

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.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.     

Genetic variation underlies temperature tolerance of embryos in the sea urchin Heliocidaris erythrogramma armigera

Ocean warming can alter natural selection on marine systems, and in many cases, the long‐term persistence of affected populations will depend on genetic adaptation. In this study, we assess the potential for adaptation in the sea urchin Heliocidaris erythrogramma armigera, an Australian endemic, that is experiencing unprecedented increases in ocean temperatures. We used a factorial breeding design to assess the level of heritable variation in larval hatching success at two temperatures. Fertilized eggs from each full‐sibling family were tested at 22 °C (current spawning temperature) and 25 °C (upper limit of predicted warming this century). Hatching success was significantly lower at higher temperatures, confirming that ocean warming is likely to exert selection on this life‐history stage. Our analyses revealed significant additive genetic variance and genotype‐by‐environment interactions underlying hatching success. Consistent with prior work, we detected significant nonadditive (sire‐by‐dam) variance in hatching success, but additionally found that these interactions were modified by temperature. Although these findings suggest the potential for genetic adaptation, any evolutionary responses are likely to be influenced (and possibly constrained) by complex genotype‐by‐environment and sire‐by‐dam interactions and will additionally depend on patterns of genetic covariation with other fitness traits.     

Spicule Formation by Isolated Micromeres of the Sea Urchin Embryo

Micromeres are isolated at the 16-cell stage from three speciesof Japanese sea urchins, Hemicentrotus pulcherrimus, Pseudocentrotusdepressus, and Anthocidaris crassispina, and are cultured insea water containing a small amount of horse serum. In all speciesused, isolated micromeres first divide unequally as they doin vivo. The pattern and number of the subsequent cleavagesare also the same as in vivo, although they are not necessarilyclear in all cases, since the border of the adjacent cells becomeinvisible at each resting stage in some batches of embryos. After cleavage, passing through the stage when the contoursof the individual cell are obscure, decendants of the isolatedmicromeres form cell aggregates similar to the group of primarymesenchyme cells in a blastula. Within such aggregates, a spicularrudiment appears which develops either into a triradiate spiculeas in normal gastrulae or into a rod. The triradiate spiculegrows into a three-dimensional skeleton which is very similarto the normal pluteus skeleton, not only in its final shapeand size including species-specific characters but in its developmentalcourse and crystallographic nature. The rod, on the other hand,develops into either a one-dimensional or two-dimensional skeleton.These skeletons probably correspond to a part of the completeskeleton.     

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.     

The dorsoventral axis is specified prior to first cleavage in the direct developing sea urchin Heliocidaris erythrogramma

Previous fate mapping studies as well as the culture of isolated blastomeres have revealed that the dorsoventral axis is specified as early as the 2-cell stage in the embryos of the direct developing echinoid, Heliocidaris erythrogramma. Normally, the first cleavage plane includes the animal-vegetal axis and bisects the embryo between future dorsal and ventral halves. Experiments were performed to establish whether the dorsoventral axis is set up prior to the first cleavage division in H. erythrogramma. Eggs were elongated and fertilized in silicone tubes of a small diameter in order to orient the cleavage spindle and thus the first plane of cell division. Following first cleavage, one of the two resulting blastomeres was then microinjected with a fluorescent cell lineage tracer dye. Fate maps were made after culturing these embryos to larval stages. The results indicate that the first cleavage division can be made to occur at virtually any angle relative to the animal-vegetal and dorsoventral axes. Therefore, the dorsoventral axis is specified prior to first cleavage. We argue that this axis resides in the unfertilized oocyte rather than being set up as a consequence of fertilization.     

Spatial patterns of variation in color and spine shape in the sea urchin Heliocidaris erythrogramma

Abstract. Here we report on the first quantitative survey of morphological variation in the sea urchin Heliocidaris erythrogramma within Western Australia and distinguish between two subspecies found to co‐occur in this region. We surveyed urchins at multiple spatial scales along the Western Australian coastline to assess variation in dermis and spine color and, using landmark‐based geometric morphometrics, spine morphology. Both color and morphology proved to be useful for separating subspecies within Western Australia. There were four major color morphs: red dermis/violet spines (56%), red/violet‐green (23%), red/green (7%), and white/green (10%). Members of the first two color morphs had bulbous spines with wide, flattened tips, a morphology that is unique to Western Australia and characteristic of H. e. armigera, and members of the latter two consistently exhibited the narrow, pointed spines typical of specimens of H. e. erythrogramma, which has a broader distribution. In Western Australia, H. e. armigera was relatively abundant both within and among sites, but H. e. erythrogramma was found only in a few localized patches. Shifts in the relative abundance of these two subspecies occurred at fine spatial scales (<5 km), although environmental correlates of these transitions were unclear. Contrary to expectations, neither dermis color nor spine morphology varied with relative wave exposure: individuals with a red dermis or thickened spine morphology occurred at most sites regardless of exposure, and while white dermis and thinner spines only occurred at high‐exposure sites, these features were not common across the majority of exposed sites. Both color morph frequencies and spine morphology remained stable within sites over the 3‐year duration of this study. While the ecological significance of this morphological variation remains unclear, the consistency of the association between color and spine morphology, occurring across fine spatial scales, suggests that strong environmental or genetic factors are involved in maintaining morphological differentiation between these two subspecies.     

Male-by-female interactions influence fertilization success and mediate the benefits of polyandry in the sea urchin Heliocidaris erythrogramma

Numerous studies have reported that females benefit from mating with multiple males (polyandry) by minimizing the probability of fertilization by genetically incompatible sperm. Few, however, have directly attributed variation in female reproductive success to the fertilizing capacity of sperm. In this study we report on two experiments that investigated the benefits of polyandry and the interacting effects of males and females at fertilization in the free-spawning Australian sea urchin Heliocidaris erythrogramma. In the first experiment we used a paired (split clutch) experimental design and compared fertilization rates within female egg clutches under polyandry (eggs exposed to the sperm from two males simultaneously) and monandry (eggs from the same female exposed to sperm from each of the same two males separately). Our analysis revealed a significant fertilization benefit of polyandry and strong interacting effects of males and females at fertilization. Further analysis of these data strongly suggested that the higher rates of fertilization in the polyandry treatment were due to an overrepresentation of fertilizations due to the most compatible male. To further explore the interacting effects of males and females at fertilization we performed a second factorial experiment in which four males were crossed with two females (in all eight combinations). In addition to confirming that fertilization success is influenced by male x female interactions, this latter experiment revealed that both sexes contributed significant variance to the observed patterns of fertilization. Taken together, these findings highlight the importance of male x female interactions at fertilization and suggest that polyandry will enable females to reduce the cost of fertilization by incompatible gametes.     

The Control of Sea Urchin Metamorphosis: Ionic Effects

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus . At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation-free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation

Sea urchin micromeres were isolated from the 16-cell stage embryos and cultured until they differentiated into spicule-forming cells. Electrophoretic analysis of proteins labeled with [³⁵S]-methionine showed that the differentiation accompanied the synthesis of five cell-specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso- and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn²⁺ -containing or serum-free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn²⁺, or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.     

Coelomogenesis during the abbreviated development of the echinoid Heliocidaris erythrogramma and the developmental origin of the echinoderm pentameral body plan

The development of the coeloms is described in an echinoid with an abbreviated larval development and shows the early morphogenesis of the coeloms of the adult stage. The development is described from images obtained by laser scanning confocal microscopy. The development in Heliocidaris erythrogramma is asymmetric with a larger left coelom forming on the larval-left side and a smaller right coelom forming on the larval-right side. The right coelom forms after the development of the left coelom is well advanced. The hydrocoele forms from the anterior part of the left coelom. The five lobes of the hydrocoele from which the pentamery of the adult derives take shape on the outer, distal wall of the anterior part of the left coelom. The hydrocoele separates from the more posterior part of the left coelom, which becomes the left posterior coelom. The lobes of the hydrocoele are named, based on the site of the connexion of the stone canal to the hydrocoele. The mouth is assumed to form by penetration through only the outer, distal wall of the hydrocoele and the ectoderm. Both larval and adult polarities are evident in this larva. A comparison with coelomogenesis in the asteroid Parvulastra exigua, which also has an abbreviated development, leads to predictions of homology between the echinoderm and chordate phyla that do not require the hypothesis of a dorsoventral inversion event in chordates.