A comparative analysis of egg provisioning using mass spectrometry during rapid life history evolution in sea urchins

A dramatic life history switch that has evolved numerous times in marine invertebrates is the transition from planktotrophic (feeding) to lecithotrophic (nonfeeding) larval development—an evolutionary tradeoff with many important developmental and ecological consequences. To attain a more comprehensive understanding of the molecular basis for this switch, we performed untargeted lipidomic and proteomic liquid chromatography‐tandem mass spectrometry on eggs and larvae from three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and the distantly related planktotroph Lytechinus variegatus. We identify numerous molecular‐level changes possibly associated with the evolution of lecithotrophy in H. erythrogramma. We find the massive lipid stores of H. erythrogramma eggs are largely composed of low‐density, diacylglycerol ether lipids that, contrary to expectations, appear to support postmetamorphic development and survivorship. Rapid premetamorphic development in this species may instead be powered by upregulated carbohydrate metabolism or triacylglycerol metabolism. We also find proteins involved in oxidative stress regulation are upregulated in H. erythrogramma eggs, and apoB‐like lipid transfer proteins may be important for echinoid oogenic nutrient provisioning. These results demonstrate how mass spectrometry can enrich our understanding of life history evolution and organismal diversity by identifying specific molecules associated with distinct life history strategies and prompt new hypotheses about how and why these adaptations evolve.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

Molecular analysis of heterochronic changes in the evolution of direct developing sea urchins

The sea urchin Heliocidaris erythrogramma is a direct developer; it progresses directly from the gastrula to the juvenile adult without forming a pluteus larva. No larval skeleton is formed by mesenchyme cells, but formation of the juvenile skeleton is accelerated. We have examined two alterations in mesenchyme cell behavior that accompany this striking change in developmental pattern. 1) Rapid cell proliferation produces 1700–2200 mesenchyme cells by mid-gastrula, compared to 30–60 primary mesenchyme cells in species with typical larval development. This change may reflect the accelerated production of adult structures in H. erythrogramma. 2) B2C2 is a monoclonal antibody that recognizes primary (Anstrom et al., 1987) and adult mesenchyme cells associated with skeleton formation in typical developers. The altered pattern of B2C2 staining in H. erythrogramma (e.g., a later initial appearance of the B2C2 antigen) suggests that H. erythrogramma has deleted part of a larval program of development and accelerated its adult program of development. These results indicate that cellular and molecular heterochronies accompany the morphological changes in H. erythrogramma development.     

Morphogenetic mechanisms of coelom formation in the direct-developing sea urchin <Emphasis Type="Italic">Heliocidaris erythrogramma</Emphasis>

Indirect development via a feeding pluteus larva represents the ancestral mode of sea urchin development. However, some sea urchin species exhibit a derived form of development, called direct development, in which features of the feeding larva are replaced by accelerated development of the adult. A major difference between these two developmental modes is the timing of the formation of the left coelom and initiation of adult development. These processes occur much earlier in developmental and absolute time in direct developers and may be underlain by changes in morphogenetic processes. In this study, we explore whether differences in the cellular mechanisms responsible for the development of the left coelom and adult structures are associated with the change in the timing of their formation in the direct-developing sea urchin Heliocidaris erythrogramma. We present evidence that left coelom formation in H. erythrogramma, which differs in major aspects of coelom formation in indirect developers, is not a result of cell division. Further, we demonstrate that subsequent development of adult structures requires cell division.     

Primary Invagination of the Vegetal Plate During Sea Urchin Gastrulation

The initial phase of echinoid gastrulation, primary invagination,involves an inpocketing of a monolayered epithelium. To gaininformation about the nature of the mechanical forces that areresponsible for primary invagination, several experimental approacheshave been taken, using the transparent embryos of the sea urchin,Lytechinus pictus, as the principal material. Vegetal platesisolated microsurgically well before the onset of gastrulationwill invaginate normally, demonstrating that the forces responsiblefor primary invagination are generated by the cells in the vegetal to of the embryo. As shown by serial reconstructions of L.pictus embryos, relatively few cells (about 100) take part inprimary invagination. Both the number of cells and the totalvolume of tissue in the wall of the archenteron increase withtime. Even so, it can be shown that very little movement ofcells over the lip of the blastopore takes place during primaryinvagination, and this process is best viewed as a simple inpocketingof the vegetal epithelium. The cells in the wall of the archenteronhave a distinctive shape; they are elongated along their apico-basalaxes and frequently have enlarged, rounded, basal ends. However,they do not undergo any dramatic changes in shape during primaryinvagination. In particular, there is only a slight decreasein the height of the cells (length along the apico-basal axis),a result that is inconsistent with the hypothesis that invaginationis due to cell rounding (Gustafson and Wolpert, 1967). Examinationof L. pictus and Strongylocentrotus purpuratus gastrulae bytransmission electron microscopy reveals that cells in the wallof the archenteron continue to be joined by typical junctionalcomplexes during primary invagination. In addition, the morphologyof the junctional complex at the gastrula stage is more elaboratethan previously described. Sparse bands of micronlaments areassociated with the plasma membrane at the level of the junctionalcomplexes in both endodermal and ectodermal cells. These andother relevant data on early echinoid gastrulation are discussedin relation to several possible mechanisms of epithelial morphogenesis.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

A cyto-embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus , which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis , even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus . These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well-known species of sea urchins.     

Local shifts in position and polarized motility drive cell rearrangement during sea urchin gastrulation

This study examines the mechanisms of epithelial cell rearrangement during archenteron elongation in the sea urchin embryo using scanning electron microscopy, differential interference contrast videomicroscopy, cell marking, and fluorescently labeled chimaeric clones. Archenteron elongation involves two major processes: local shifts in position of cells in the archenteron wall and polarized motility of the cells as they rearrange. Fluorescently labeled chimaeric clones introduced into the archenteron of Lytechinus pictus are initially 4-5 cells wide; by the end of gastrulation the clones elongate and narrow, so that they are one cell wide in the narrowest region of the archenteron. The extent of clonal mixing indicates that cells in the archenteron change their relative positions by only 1-2 cell diameters during cell rearrangement. Cells at the blastopore rearrange concomitantly with cells in the archenteron, resulting in a 35% decrease in blastopore diameter. Endoderm cells undergo polarized, stage-specific changes in shape and motility as they rearrange; (1) they flatten markedly along their apical-basal axis throughout archenteron elongation; (2) just prior to the onset of cell rearrangement, basal surfaces of all cells in the archenteron extend long, polarized lamellipodial protrusions along the axis of extension of the archenteron; (3) as cell rearrangement begins, basal surfaces round up and the cells become isodiametric; (4) by the 3/4 gastrula stage the cells become stretched along the animal-vegetal axis, apparently due to filopodial traction, and finally (5) they continue to rearrange, returning to a less elongated shape by the end of gastrulation. Direct observation of gastrulation in the cidaroid Eucidaris tribuloides indicates that in this species cell rearrangement is accomplished by progressive circumferential intercalation of cells without upwardly directed filopodia. This intercalation is accompanied by explosive, apparently stochastic, cortical blebbing activity at the boundaries between cells, suggesting that in addition to whatever cell rearrangement may be generated by filopodial tension, such activity is an important component of the active rearrangement process.     

Dynamic regulation of LARG in blastopore closure and archenteron formation during Xenopus laevis gastrulation

Rho GTPases have important roles in regulating cell migration and are activated by Rho-specific guanine nucleotide exchange factors (RhoGEFs). However, the role of leukemia-associated RhoGEF (LARG), responding to G12/13 family, has not been studied in vertebrate development. Here, the in vivo biochemical function of LARG was examined during early embryonic development in African frog Xenopus laevis. Gain-of-function study was performed by injecting the RNA of full-length xLARG to 2 cell-stage embryos. The ectopic expression of this protein resulted in the defect of blastopore closure during early embryogenesis. Expression of the dominant-negative form caused the defect in cell movement and following archenteron formation during late gastrulation, which is represented by the blister formation in the ventral side of the embryos. The phenotype was rescued by co-expressing the mutant with Rho or wild type xLARG, confirming the specificity of the dominant-negative activity of xLARG mutant. In this study, I showed for the first time that the spatiotemporal expression of xLARG is very dynamic and specifically regulated in early Xenopus embryonic development and xLARG may mediate Gα₁₃ signal to activate Rho to exert its function in gastrulation movement and archenteron formation. My results implicate that the dynamic regulation of maternal and zygotic xLARG expression and its biochemical activity is necessary for proper gastrulation.     

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.     

MORE THAN BINDIN DIVERGENCE: REPRODUCTIVE ISOLATION BETWEEN SYMPATRIC SUBSPECIES OF A SEA URCHIN BY ASYNCHRONOUS SPAWNING

The evolution of reproductive barriers is crucial to the process of speciation. In the Echinoidea, studies have focused on divergence in the gamete recognition protein, bindin, as the primary isolating mechanism among species. As such, the capacity of alternate mechanisms to be effective reproductive barriers and the phylogenetic context in which they arise is unclear. Here, we examine the evolutionary histories and factors limiting gene exchange between two subspecies of Heliocidaris erythrogramma that occur sympatrically in Western Australia. We found low, but significant differentiation between the subspecies in two mitochondrial genes. Further, coalescent analyses suggest that they diverged in isolation on the east and west coasts of Australia, with a subsequent range expansion of H. e. erythrogramma into Western Australia. Differentiation in bindin was minimal, indicating gamete incompatibility is an unlikely reproductive barrier. We did, however, detect strong asynchrony in spawning seasons; H. e. erythrogramma spawned over summer whereas H. e. armigera spawned in autumn. Taken together, we provide compelling evidence for a recent divergence of these subspecies and their reproductive isolation without gamete incompatibility. Western Australian H. erythrogramma may therefore present an intriguing case of incipient speciation, which depends on long‐term persistence of the factors underlying this spawning asynchrony.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation

Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin protein Daam1 and an effector molecule XProfilin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProfilin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProfilin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1 regulates blastopore closure, overexpression or depletion of XProfilin2 specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1. Together, our studies demonstrate that XProfilin2 and XProfilin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.     

Molecular phylogeny of echinometrid sea urchins: more species of Heliocidaris with derived modes of reproduction

Abstract. Pachechinus bajulus is an endemic Australian sea urchin with an unusual mode of brooded larval development. We used mitochondrial and nuclear gene sequences to estimate the phylogenetic relationships among Pachechinus and other Echinometridae, including well‐studied species of Heliocidaris with planktonic development. We found strong evidence for the planktotrophic species Heliocidaris tuberculata as the sister group to a clade of three closely related species in which development is known (Heliocidaris erythrogramma, P. bajulus) or suspected (Pachechinus australiae) to be lecithotrophic. Clade support values and likelihood ratio tests rejected monophyly of Heliocidaris species. The sister group to H. erythrogramma is most likely the two Pachechinus species. We resolve the paraphyly problem by reassigning the Pachechinus species to the genus Heliocidaris (the senior synonym), which has six extant species including Heliocidaris australiae and Heliocidaris bajulus. The phylogeny has potentially important implications for comparative studies of developmental morphology and genetics that have assumed a close sister‐group relationship between H. erythrogramma and H. tuberculata, and highlights the need for such data from H. bajulus and other Heliocidaris species.     

An SEM study of cellular morphology,contact, and arrangement,as related to gastrulation inXenopus laevis

Summary Cellular morphology, contact, and arrangement in the late blastula and in various stages of gastrulation ofXenopus were examined by SEM of specimens dissected after fixation or fractured in amyl acetate. The prospective ectoderm of the blastocoel roof consists of several layers of interdigitating cells connected by numerous small protrusions which may function in the decrease in number of cell layers observed during ectodermal epiboly. During gastrulation, prospective mesoderm is regionally differentiated by cellular morphology and arrangement into preinvolution mesoderm, the mesodermal involution zone, and involuted mesoderm. The involuted anterodorsal (head), lateral, and ventral mesoderm consists of a stream of loosely-packed, irregularly shaped cells having large extensions of the cell body attached locally to other cells by small protrusions. Involuted posterodorsal mesoderm (chordamesoderm) consists of elongated cells arranged in palisade fashion and connected by similar protrusions. Involuted mesodermal cells in all regions are attached to the overlying prospective ectodermal cells by numerous small protrusions along the entire interface between the two cell layers. Suprablastoporal endodermal cells involute as an epithelial sheet, changing in shape in the process, to form the roof of the archenteron. Bottle cell morphology, arrangement, and position with respect to the mesodermal cell stream is described. Evidence presented here and elsewhere suggests that involution of mesoderm and of the archenteron roof inXenopus is dependent primarily upon the relative movement of the mesodermal cell stream and of the overlying ectoderm.     

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.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Morphogenesis and organogenesis in the regenerating planktotrophic larvae of asteroids and echinoids

In a previous study, we described complete body regeneration (with organogenesis) following surgical bisection in the planktotrophic larvae of the asteroids Luidia foliolata and Pisaster ochraceus. Here we present further detailed observations of these unique regenerative processes not presented in the previous paper. Furthermore, we describe for the first time complete regeneration following surgical bisection of planktotrophic larvae of the regular echinoid Lytechinus variegatus and the irregular echinoid Dendraster excentricus. Larvae of both asteroids and echinoids displayed a capacity for rapid regeneration regardless of their developmental stage. Within 48 h after bisection, aggregations of mesenchyme cells with pseudopodia were observed at the site of surgical bisection. These cellular aggregations were similar in appearance to the mesenchymal blastemas that form in adult echinoderms prior to their arm regeneration, and to those described in other deuterostomes that undergo regeneration. When asteroid larvae were surgically bisected in the early stages of their development, clusters of mesenchyme cells developed into completely new pairs of coelomic pouches located anterior to the newly regenerated digestive tract. This indicates that cell fate in regenerating asteroid larvae remains indeterminate during early development. In the larvae of P. ochraceus, regardless of the developmental stage at the time of bisection, both the anterior and posterior portions regenerated all their missing organs and tissues. However, the larvae of L. foliolata displayed differential regenerative capacity in bisected larval halves at the late bipinnaria stage. The differences observed may be due to differences in larval development (L. foliolata has no brachiolaria stage), and may have evolutionary implications. In the regular echinoid L. variegatus, both larval portions regenerated into morphologically and functionally normal larvae that were indistinguishable from non-bisected control larvae. The regenerative processes were similar to those we observed in planktotrophic asteroid larvae. Regenerating larvae readily metamorphosed into normal juveniles. In the irregular echinoid D. excentricus, posterior portions of larvae completed regeneration and metamorphosis, but anterior portions regenerated only partially during the 2-week study. Our observations confirm that asteroid and echinoid larvae provide excellent models for studies of regeneration in deuterostomes.