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Gastrulation in the sea urchin begins with ingression of the primary mesenchyme cells (PMCs) at the vegetal pole of the embryo. After entering the blastocoel the PMCs migrate, form a syncitium, and synthesize the skeleton of the embryo. Several hours after the PMCs ingress the vegetal plate buckles to initiate invagination of the archenteron. That morphogenetic process occurs in several steps. The nonskeletogenic cells produce the initial inbending of the vegetal plate. Endoderm cells then rearrange and extend the length of the gut across the blastocoel to a target near the animal pole. Finally, cells that will form part of the midgut and hindgut are added to complete gastrulation. Later, the stomodeum invaginates from the oral ectoderm and fuses with the foregut to complete the archenteron. In advance of, and during these morphogenetic events, an increasingly complex input of transcription factors controls the specification and the cell biological events that conduct the gastrulation movements. WIREs Dev Biol2012, 1:231–252. doi: 10.1002/wdev.18 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and GenomicsEarly Embryonic Development > Gastrulation and NeurulationComparative Development and Evolution > Model Systems

Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm.

Secondary axis specification is a process that relies on asymmetric nuclearization of transcription factors in flies and vertebrates, such that the crucial factor is nuclear and therefore functional only in cells along one side of the embryo. In vertebrates, this transcription factor is β-catenin, which is canonically activated downstream from Wnt signals. However, the sea urchin uses asymmetric β-catenin nuclearization during specification of the primary animal-vegetal axis, rather than the secondary oral-aboral (OA) axis. OA specification relies instead on the asymmetric localization of p38 MAPK, a signaling kinase that directly modulates transcription factor activity. A number of genes are expressed in the oral territory downstream from p38, including Nodal and Goosecoid, both of which are associated with secondary axis specification in vertebrate embryos. Because the p38 asymmetry is the earliest known event in the specification of the OA axis, an outstanding question concerns identifying the apparatus upstream from p38 that regulates its asymmetrical activity. Intriguingly, this may be controlled by reactive oxygen species released from the mitochondria, which are asymmetrically distributed about the OA axis.

p38 is a MAPK that has been shown to induce a wide variety of biological effects in cell culture,in response to a wide range of stimuli. These effects are dependent not only on the stimuli, butalso on the cellular context, resulting in a bewildering array of possibilites1. For example, p38was shown to induce apoptosis in some cells, but prevent apoptosis in others2. Similarly opposedeffects had been observed with respect to cell cycle regulation2. The role of p38 in inflammatorydisease has been appreciated from the beginning, since it was initially identified as an cytokineinducer3. More recently, p38 function has been evaluated in vivo, and through these studies p38has emerged as an important regulator of both embryonic development and cancer progression.This review will focus on these in vivo studies in an effort to provide perspective on p38biologically and as a pharmacological target.

T-box family transcription factors have been identified in many organisms and are frequently associated with patterning events during embryonic development. With an interest in the molecular basis of patterning in the sea urchin embryo, we identified several members of the T-box family inLytechinus variegatus. Here, we report the cloning and characterization of an ortholog of the Tbx2/3 subfamily, LvTbx2/3. To characterize the spatial distribution of LvTbx2/3 protein throughout sea urchin embryogenesis, a polyclonal antiserum was generated. Nuclear localization of LvTbx2/3 initiated at the mesenchyme blastula stage and protein was present into the pluteus stage. Localization was asymmetric throughout this period and costaining with marker genes indicated that asymmetry was about the oral/aboral (O/A) axis. Asymmetric distribution of LvTbx2/3 was observed in the aboral territories of all three germ layers. In the skeletogenic mesoderm lineage, LvTbx2/3 expression was dynamic because expression appeared initially in all skeletogenic mesenchyme cells (PMCs) but,subsequently, became refined solely to the aboral ones during skeletogenesis. To determine if the aboral expression of LvTbx2/3 is linked between germ layers, and to place LvTbx2/3 in the sequence of events that specifies the O/A axis, the effects of a series of perturbations to O/A polarity on LvTbx2/3 expression in each germ layer were examined. Preventing the nuclear localization of β-catenin, pharmacological disruption of the O/A axis with NiCl2, overexpression ofBMP2/4 and disruption of the extracellular matrix all blockedLvTbx2/3 expression in all germ layers. This indicates that expression of LvTbx2/3 in the aboral territories of each germ layer is a common aspect of O/A specification, downstream of the molecular events that specify the axis. Furthermore, blocking the nuclear localization ofβ-catenin, overexpression of BMP2/4 and disruption of the extracellular matrix also prevented the oral (stomodael) expression of LvBrachyury (LvBrac) protein, indicating that the O/A axis is established by a complex series of events. Last, the function of LvTbx2/3 in the formation of the O/A axis was characterized by examining the phenotypic consequences of ectopic expression of LvTbx2/3 mRNA on embryonic development and the expression of marker genes that identify specific germ layers and tissues. Ectopic expression of LvTbx2/3 produced profound morphogenetic defects in derivatives of each germ layer with no apparent loss in specification events in those tissues. This indicates that LvTbx2/3 functions as a regulator of morphogenetic movements in the aboral compartments of the ectoderm, endoderm and mesoderm.

The studies described here sought to identify and characterize genes involved in the gastrulation and morphogenetic movements that occur during sea urchin embryogenesis. An orthologue of the T-box family transcription factor, Brachyury, was cloned through a candidate gene approach. Brachyury(T) is the founding member of this T-box transcription factor family and has been implicated in gastrulation movements in Xenopus, zebrafish, and mouse embryogenesis. Polyclonal serum was generated to LvBrac in order to characterize protein expression. LvBrac initially appears at mesenchyme blastula stage in two distinct regions with embryonic expression perduring until pluteus stage. Vegetally, LvBrac expression is in endoderm and lies circumferentially around the blastopore. This torus-shaped area of LvBrac expression remains constant in size as endoderm cells express LvBrac upon moving into that circumference and cease LvBrac expression as they leave the circumference. Vegetal expression remains around the anus through pluteus stage. The second domain of LvBrac expression first appears broadly in the oral ectoderm at mesenchyme blastula stage and at later embryonic stages is refined to just the stomodael opening. Vegetal LvBrac expression depends on autonomous β-catenin signaling in macromeres and does not require micromere or veg2-inductive signals. It was then determined that LvBrac is necessary for the morphogenetic movements occurring in both expression regions. A dominant-interfering construct was generated by fusing the DNA binding domain of LvBrac to the transcriptional repression module of the DrosophilaEngrailed gene in order to perturb gene function. Microinjection of mRNA encoding this LvBrac-EN construct resulted in a block in gastrulation movements but not expression of endoderm and mesoderm marker genes. Furthermore, injection of LvBrac-EN into one of two blastomeres resulted in normal gastrulation movements of tissues derived from the injected blastomere, indicating that LvBrac downstream function may be nonautonomous during sea urchin gastrulation.

The molecular mechanisms guiding the positioning of the ectoderm-endoderm boundary along the animal-vegetal axis of the sea urchin embryo remain largely unknown. We report here a role for the sea urchin homolog of the Notch receptor, LvNotch, in mediating the position of this boundary. Overexpression of an activated form of LvNotch throughout the embryo shifts the ectoderm-endoderm boundary more animally along the animal-vegetal axis, whereas expression of a dominant negative form shifts the border vegetally. Mosaic experiments that target activated and dominant negative forms of LvNotch into individual blastomeres of the early embryo, combined with lineage analyses, further reveal that LvNotch signaling mediates the position of this boundary by distinct mechanisms within the animal versus vegetal portions of the embryo. In the animal region of the embryo, LvNotch signaling acts cell autonomously to promote endoderm formation more animally, while in the vegetal portion, LvNotch signaling also promotes the ectoderm-endoderm boundary more animally, but through a cell non-autonomous mechanism. We further demonstrate that vegetal LvNotch signaling controls the localization of nuclear β-catenin at the ectoderm-endoderm boundary. Based on these results, we propose that LvNotch signaling promotes the position of the ectoderm-endoderm boundary more animally via two mechanisms: (1) a cell-autonomous function within the animal region of the embryo, and (2) a cell non-autonomous role in the vegetal region that regulates a signal(s) mediating ectoderm-endoderm position, possibly through the control of nuclear β-catenin at the boundary.

At fourth cleavage of sea urchin embryos four micromeres at the vegetal pole separate from four macromeres just above them in an unequal cleavage. The micromeres have the capacity to induce a second axis if transplanted to the animal pole and the absence of micromeres at the vegetal pole results in the failure of macromere progeny to specify secondary mesenchyme cells (SMCs). This suggests that micromeres have the capacity to induce SMCs. We demonstrate that micromeres require nuclear β-catenin to exhibit SMC induction activity. Transplantation studies show that much of the vegetal hemisphere is competent to receive the induction signal. The micromeres induce SMCs, most likely through direct contact with macromere progeny, or at most a cell diameter away. The induction is quantitative in that more SMCs are induced by four micromeres than by one. Temporal studies show that the induction signal is passed from the micromeres to macromere progeny between the eighth and tenth cleavage. If micromeres are removed from hosts at the fourth cleavage, SMC induction in hosts is rescued if they later receive transplanted micromeres between the eighth and tenth cleavage. After the tenth cleavage addition of induction-competent micromeres to micromereless embryos fails to specify SMCs. For macromere progeny to be competent to receive the micromere induction signal, β-catenin must enter macromere nuclei. The macromere progeny receive the micromere induction signal through the Notch receptor. Signaling-competent micromeres fail to induce SMCs if macromeres express dominant-negative Notch. Expression of an activated Notch construct in macromeres rescues SMC specification in the absence of induction-competent micromeres. These data are consistent with a model whereby β-catenin enters the nuclei of micromeres and, as a consequence, the micromeres produce an inductive ligand. Between the eighth and tenth cleavage micromeres induce SMCs through Notch. In order to be receptive to the micromere inductive signal the macromeres first must transport β-catenin to their nuclei, and as one consequence the Notch pathway becomes competent to receive the micromere induction signal, and to transduce that signal. As Notch is maternally expressed in macromeres, additional components must be downstream of nuclear β-catenin in macromeres for these cells to receive and transduce the micromere induction signal.

To examine whether a BMP signaling pathway functions in specification of cell fates in sea urchin embryos, we have cloned sea urchin BMP2/4, analyzed its expression in time and space in developing embryos and assayed the developmental consequences of changing its concentration through mRNA injection experiments. These studies show that BMP4 mRNAs accumulate transiently during blastula stages, beginning around the 200-cell stage, 14 hours postfertilization. Soon after the hatching blastula stage, BMP2/4 transcripts can be detected in presumptive ectoderm, where they are enriched on the oral side. Injection of BMP2/4 mRNA at the one-cell stage causes a dose-dependent suppression of commitment of cells to vegetal fates and ectoderm differentiates almost exclusively as a squamous epithelial tissue. In contrast, NOGGIN, an antagonist of BMP2/4, enhances differentiation of endoderm, a vegetal tissue, and promotes differentiation of cells characteristic of the ciliated band, which contains neurogenic ectoderm. These findings support a model in which the balance of BMP2/4 signals produced by animal cell progeny and opposing vegetalizing signals sent during cleavage stages regulate the position of the ectoderm/ endoderm boundary. In addition, BMP2/4 levels influence the decision within ectoderm between epidermal and nonepidermal differentiation.

Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm.

At gastrulation in the sea urchin embryo dramatic cell adhesion changes contribute to primary mesenchyme cell ingression movements and to cell rearrangements during archenteron invagination. At ingression, quantitative adhesion assays demonstrated previously that primary mesenchyme cells (PMCs) change their affinity for neighboring cells, for a fibronectin-like substrate, and for the hyaline layer. To investigate the molecular basis for these and other differential cell affinities at gastrulation, we have identified an integrin that appears to be responsible for specific alterations in cell–substrate adhesion to laminin. During early cleavage stages blastomeres adhere poorly to laminin substrates. Around hatching there is a large increase in the ability of blastomeres to bind to laminin and this increase correlates temporally with the expression of an integrin on the basal surface all blastomeres. PMCs, after undergoing their epithelial–mesenchymal transition, have a strongly reduced affinity for laminin relative to ectoderm cells and, correspondingly, do not stain for the presence of the integrin. We identified the α integrin cDNA fromLytechinus variegatusby RT-PCR. Overlapping clones were obtained from a midgastrula cDNA library to provide a complete sequence for the integrin. The composite cDNA encoded a protein that was most similar to the α5 subgroup of vertebrate integrins, but there was not a definitive vertebrate integrin homolog. Northern blots and Western immunoblots showed that the sea urchin integrin, which we have named αSU2, is present in eggs and during all stages of development. Immunolocalization with specific polyclonal antibodies showed that αSU2 first appears on the basal cell surface of epithelia at the midblastula stage, at a time correlating with the increase in adhesive affinity for laminin. The protein remains at high levels on the basal surface of ectoderm cells but is temporarily reduced or eliminated from endoderm cells during their convergent–extension movements. To confirm integrin binding specificity, αSU2 was transfected into an α-integrin-deficient CHO cell line. αSU2-expressing CHO cells bound well to isolated sea urchin basal lamina and to purified laminin. The transfected cells bound weakly or not at all to fibronectin, type I collagen, and type IV collagen. This is consistent with the hypothesis that αSU2 integrin functions by binding epithelial cells to laminin in the basal lamina.In vivo,modulation of αSU2 integrin expression correlates with critical adhesive changes during cleavage and gastrulation. Thus, this protein appears to be an important contributor to the morphogenetic rearrangements that characterize gastrulation in the sea urchin embryo.

Cell-cell interactions are thought to regulate the differential specification of secondary mesenchyme cells (SMCs) and endoderm in the sea urchin embryo. The molecular bases of these interactions, however, are unknown. We have previously shown that the sea urchin homologue of the LIN-12/Notch receptor, LvNotch, displays dynamic patterns of expression within both the presumptive SMCs and endoderm during the blastula stage, the time at which these two cell types are thought to be differentially specified (Sherwood, D. R. and McClay, D. R. (1997) Development 124, 3363-3374). The LIN-12/Notch signaling pathway has been shown to mediate the segregation of numerous cell types in both invertebrate and vertebrate embryos. To directly examine whether LvNotch signaling has a role in the differential specification of SMCs and endoderm, we have overexpressed activated and dominant negative forms of LvNotch during early sea urchin development. We show that activation of LvNotch signaling increases SMC specification, while loss or reduction of LvNotch signaling eliminates or significantly decreases SMC specification. Furthermore, results from a mosaic analysis of LvNotch function as well as endogenous LvNotch expression strongly suggest that LvNotch signaling acts autonomously within the presumptive SMCs to mediate SMC specification. Finally, we demonstrate that the expansion of SMCs seen with activation of LvNotch signaling comes at the expense of presumptive endoderm cells, while loss of SMC specification results in the endoderm expanding into territory where SMCs usually arise. Taken together, these results offer compelling evidence that LvNotch signaling directly specifies the SMC fate, and that this signaling is critical for the differential specification of SMCs and endoderm in the sea urchin embryo.

β-catenin is thought to mediate cell fate specification events by localizing to the nucleus where it modulates gene expression. To ask whether β-catenin is involved in cell fate specification during sea urchin embryogenesis, we analyzed the distribution of nuclear β-catenin in both normal and experimentally manipulated embryos. In unperturbed embryos, β-catenin accumulates in nuclei that include the precursors of the endoderm and mesoderm, suggesting that it plays a role in vegetal specification. Using pharmacological, embryological and molecular approaches, we determined the function of β-catenin in vegetal development by examining the relationship between the pattern of nuclear β-catenin and the formation of endodermal and mesodermal tissues. Treatment of embryos with LiCl, a known vegetalizing agent, caused both an enhancement in the levels of nuclear β-catenin and an expansion in the pattern of nuclear β-catenin that coincided with an increase in endoderm and mesoderm. Conversely, overexpression of a sea urchin cadherin blocked the accumulation of nuclear β-catenin and consequently inhibited the formation of endodermal and mesodermal tissues including micromere-derived skeletogenic mesenchyme. In addition, nuclear β-catenin-deficient micromeres failed to induce a secondary axis when transplanted to the animal pole of uninjected host embryos, indicating that nuclear β-catenin also plays a role in the production of micromere-derived signals. To examine further the relationship between nuclear β-catenin in vegetal nuclei and micromere signaling, we performed both transplantations and deletions of micromeres at the 16-cell stage and demonstrated that the accumulation of β-catenin in vegetal nuclei does not require micromere-derived cues. Moreover, we demonstrate that cell autonomous signals appear to regulate the pattern of nuclear β-catenin since dissociated blastomeres possessed nuclear β-catenin in approximately the same proportion as that seen in intact embryos. Together, these data show that the accumulation of β-catenin in nuclei of vegetal cells is regulated cell autonomously and that this localization is required for the establishment of all vegetal cell fates and the production of micromere-derived signals.

After fertilization most eggs become surrounded by a complex extracellular matrix. This study examines those matrix assembly processes that are triggered by fertilization of the sea urchin egg. The study uses antibodies that identify five different storage compartments in the egg. These compartments release their protein contents in a highly regulated fashion to assemble and modify the extraembryonic layers. The exocytosis sequence begins with a fertilization wave that progresses from the site of sperm entry and elevates the fertilization envelope above a water-filled perivitelline space. The immediate surface of the zygote then becomes covered by a newly secreted hyaline layer. Prior to fertilization some of the antigens are localized to cortical granules. Others are found in “basal laminar vesicles” that are released in a wave beginning at about 30 sec, or roughly at the same time as cortical granule exocytosis. The remaining antigens are exocytosed with a rather precise timing, but with a delay of several to tens of minutes relative to the first wave of exocytosis. “Apical vesicles,” so named because antigens from this class are preferentially exocytosed toward the apical cell surface of polarized cells, include antigens that are exocytosed beginning at about 5 min postfertilization. The fourth compartment, named “echinonectin vesicles” release echinonectin, a protein that is deposited to the inner side of the hyaline layer. Surface staining of echinonectin is first detected about 10–15 min following sperm contact. Finally, maternal cadherin, which is stored in yet a fifth distinct compartment, is not detected on the surface until at least 30 min following fertilization. The data are also consistent with the notion that the tightly regulated timing of exocytosis contributes to the ordered assembly of the hyaline layer and elevation of the fertilization envelope. Finally, two of the vesicle classes continue to exocytose after the cells become polarized. In polarized cells apical and basal laminar antigens are trafficked toward opposite sides of the same cell after passing through the sametrans-Golgi network-like compartment.

In the sea urchin embryo the primary mesenchyme cells (PMCs) produce a CaCO3skeleton in a pattern that is species specific. To establish whether skeletal pattern is specified autonomously by the PMCs or through instructive interactions with the ectoderm, we have taken advantage of improvements in cell transplantation techniques to produce PMC/ectoderm chimeras between two species. It was found that the pattern of the skeletons produced in these chimeric embryos depended upon the source of the PMCs. Thus, skeletal pattern appears to be specified autonomously by the PMCs. In comparing this data with other information known about skeletogenesis, it is proposed that the ectoderm provides spatial and temporal information for the correct placement and timing of skeletogenesis, and that the PMCs then synthesize the skeleton according to an endogenous program. The ability of PMCs from one species to produce an appropriately patterned skeleton in the ectoderm of another indicates that the cues provided by the ectoderm are largely conserved between species.

Paracentrotus lividussea urchin nectin (Pl-nectin) is an extracellular matrix (ECM) protein of the sea urchin embryo on the apical surface of the ectoderm and has been shown to be an adhesive substrate for embryonic cells. A monoclonal antibody (McAb) toPl-nectin was generated that inhibits the adhesion of blastula cells toPl-nectin-coated substrates in anin vitrofunctional assay. To examine for possiblein vivofunctions ofPl-nectin, Fab fragments (Fabs) ofPl-nectin McAb were added to early blastulae. Ingression of primary mesenchyme cells was not affected by Fabs. As control embryos reached the pluteus stage, treated embryos showed a severe inhibition of skeletal elongation and patterning. When the Fabs were injected directly into the blastocoel, even at higher concentration than was applied externally, skeletogenesis was normal. Therefore, the effect of the antibody on spiculogenesis was indirect. The treatment was partially reversible as embryos eventually seemed to recover and elongate spicules, although with an incorrect patterning. Migration of pigment cells was also affected by the Fabs, since they did not disperse throughout the ectoderm but remained clustered in ectopic areas. In contrast, the development of endoderm structures was not affected. Our results indicate that in the sea urchin embryo the appropriate contact of ectodermal cells with outer ECM components is essential for the correct morphogenesis of inner mesodermal structures.

By comparing the spatial and temporal distribution of three proteins during early development in seven echinoid species, we demonstrate that both heterochronies and heterotopies in gene‐product expression have accompanied the radiation of post‐Paleozoic echinoids. All three proteins examined showed significant alterations in time of expression, site of expression, or both. These molecular heterochronies and heterotopies indicate that early development is not necessarily as evolutionarily conservative as morphology of embryos alone would suggest. Evolutionary alterations in early development may be more common than is generally assumed.

Few treatments are known that perturb the dorsoventral axis of the sea urchin embryo. We report here that the dorsoventral polarity of the sea urchin embryo can be disrupted by treatment of embryos with NiCl2. Lytechinus variegatus embryos treated with 0.5 mM NiCl2 from fertilization until the early gastrula stage appear morphologically normal until the midgastrula stage, when they fail to acquire the overt dorsoventral polarity characteristic of untreated siblings. The ectoderm of normal embryos possesses two ventrolateral thickenings just above the vegetal plate region. In nickel-treated embryos, these become expanded as a circumferential belt around the vegetal plate. The ectoderm just ventral to the animal pole normally invaginates to form a stomodeum, which then fuses with the tip of the archenteron to produce the mouth. In nickeltreated embryos, the stomodeal invagination is expanded to become a circumferential constriction, and it eventually pinches off as the tip of the archenteron fuses with it to produce a mouth. Primary mesenchyme cells form a ring in the lateral ectoderm, but as many as a dozen spicule rudiments can form in a radial pattern. Dorsoventral differentiation of ectodermal tissues is profoundly perturbed: nickel-treated embryos underexpress transcripts of the dorsal (aboral) gene LvS1, they overexpress the ventral (oral) ectodermal gene product, EctoV, and the ciliated band is shifted to the vegetal margin of the embryo. Although some dorsoventral abnormalities are observed, animal-vegetal differentiation of the archenteron and associated structures seems largely normal, based on the localization of region-specific gene products. Gross differentiation of primary mesenchyme cells seems unaffected, since nickel-treated embryos possess the normal number of these cells. Furthermore, when all primary mesenchyme cells are removed from nickel-treated embryos, some secondary mesenchyme cells undergo the process of “conversion” (Ettensohn, C. A. and McClay, D. R. (1988) Dev. Biol. 125, 396-409), migrating to sites where the larval skeleton would ordinarily form and subsequently producing spicule rudiments. However, the skeletal pattern formed by the converted cells is completely radialized. Our data suggest that a major effect of NiCl2 is to alter commitment of ectodermal cells along the dorsoventral axis. Among the consequences appears to be a disruption of pattern formation by mesenchyme cells.

Although much is known about the specification and determination of the two primary axes (animal/vegetal and dorsoventral or oral/aboral) in a number of embryos, little is understood about bilaterality. In the sea urchin, left/right asymmetry is crucial to normal development as the echinus or adult rudiment is positioned on the left side of the larva. We examined the establishment of bilateral asymmetry in Lytechnis variegatus embryos by determining the relationship of the first cleavage planes to the left/right axis. Embryos were bisected at different times to determine when the bilateral axis is committed. These lineage tracing and cell separation experiments demonstrated that the first cleavage plane divides the embryo into left and right halves, although this is conditional until after late blastula stage. The relationship between the specification of the dorsoventral axis and the bilateral axis was examined experimentally. In other species when the dorsal and ventral halves of early echinoderm embryos (preblastula) are separated, the dorsal half often reverses (180°) its dorsoventral axis. We asked whether those larvae with an inverted dorsoventral axis would shift the position of the echinus rudiment from the original left side to the new left side. If so, it would demonstrate that the larval asymmetry is dependent upon specification of the dorsoventral axis. Using a combination of lineage tracing and cell separation techniques, we show that the left/right asymmetry is specified with respect to the dorsoventral axis.

The hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development. The major constituent of the hyaline layer is the protein hyalin, a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Here we provide a molecular characterization of hyalin and identify a region of the protein that is important for its function in cell adhesion. Partial hyalin cDNAs were identified from two sea urchin species,Strongylocentrotus purpuratusandLytechinus variegatus,by screening expression libraries with monoclonal antibodies to hyalin. The cDNAs each encode a tandemly arranged series of conserved repeats averaging 84 amino acids. These hyalin repeats are as similar between the two species as they are to repeats within each species, suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences. The hyalin mRNA is approximately 12 kb in length and is present in developing oocytes coincident with the appearance of cortical granules, the vesicle in which the hyalin protein is specifically packaged. The mRNA is present throughout oogenesis but is rapidly lost at oocyte maturation so that eggs and early embryos have no detectable hyalin mRNA. The hyalin protein, however, remains at relatively constant levels throughout development. Thus, all the hyalin protein present during early development, when no RNA is detectable, is maternally derived and exocytosed from cortical granules at fertilization. Hyalin mRNA reaccumulates in embryos beginning at the mesenchyme blastula stage; a RNA gel blot andin situhybridization analysis of gastrulae and larvae shows a progressive confinement of hyalin mRNA to the aboral ectoderm. Recombinant hyalin containing the tandem repeat region of the protein was expressed in bacteria and is shown to serve as an adhesive substrate, almost equal to that of native hyalin, in cell adhesion assays. This adhesive activity is partially blocked by dilute hyalin monoclonal antibody Tg-HYL to the same extent as that for native hyalin. Thus, this hyalin repeat region appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development and now enable investigators to focus on the mechanisms of cell interactions with the hyaline layer.

β-Catenin was originally identified biochemically as a protein that binds E-cadherin in cultured cells and that interaction was later shown to be essential for cadherin function. Independently,armadillo,the β-catenin homolog inDrosophila melanogaster,was identified as a segment polarity gene necessary for the transduction ofwingless (Wnt)signals during embryonic and larval development. Recently, several investigations have also shown that β-catenin plays a critical role in axial patterning of earlyXenopus,zebrafish, and mouse embryos. In these systems, the localization of β-catenin to the plasma membrane, cytosol, and nucleus is predictive of its role in cell adhesion and signaling. In order to examine the potential role of β-catenin in regulating cell adhesion during embryogenesis, we cloned β-catenin in the sea urchinLytechinus variegatusand characterized its subcellular distribution in cells undergoing morphogenetic movements. Indicative of a role in the establishment and maintenance of cell adhesion, β-catenin is associated with lateral cell–cell contacts and accumulates at adherens junctions from cleavage stages onward. At gastrulation, changes in junctional β-catenin localization accompany several morphogenetic events. The epithelial–mesenchymal conversion that characterizes the ingression of both primary and secondary mesenchyme cells coincides with a rapid and dramatic loss of adherens junction-associated β-catenin. In addition, epithelial cells in the archenteron display a significant decrease in adherens junction-associated β-catenin levels as they undergo convergent–extension movements. These data are consistent with a role for β-catenin in regulating cell adhesion and adherens junction function during gastrulation in the sea urchin embryo.

During development, the modulation of cadherin adhesive function is proposed to control various morphogenetic events including epithelial–mesenchymal conversions and tubulogenesis, although the mechanisms responsible for regulating cadherin activity during these events remain unclear. In order to gain insights into the regulation of cadherin function during morphogenesis, we utilized the sea urchin embryo as a model system to study the regulation of cadherin localization during epithelial–mesenchymal conversion and convergent–extension movements. Polyclonal antibodies raised against the cytoplasmic domain of a cloned sea urchin cadherin recognize three major polypeptides ofMr320, 140, and 125 kDa and specifically stain adherens junctions, and to a lesser extent, lateral membrane domains in all epithelial tissues of the embryo. Analysis of embryos during gastrulation demonstrates that changes in cadherin localization are observed in cells undergoing an epithelial–mesenchymal conversion. Ingression of primary mesenchyme cells is accompanied by the rapid loss of junctional cadherin staining and the coincident accumulation of cadherin in intracellular organelles. These data are consistent with the idea that the deadhesion of mesenchymal cells from neighboring epithelial cells involves the regulated endocytosis of cell surface cadherin molecules. Conversely, neither cadherin abundance nor localization is altered in cells of the gut which undergo convergent–extension movements during the formation of the archenteron. This observation indicates that these movements do not require the loss of junctional cadherin molecules. Instead, the necessary balance between adhesion and motility may be achieved by regulating the expression of different subtypes of cadherin molecules or modifying interactions between cadherins and catenins, proteins that bind the cytoplasmic domain of cadherin and are necessary for cadherin adhesive function. To address cadherin function at the molecular level, we used a partial cDNA representing the conserved cytoplasmic domain to identify a novel cadherin molecule in the sea urchinLytechinus variegatus.The deduced amino acid sequence of LvG-cadherin (for Goliath-cadherin) predicts that it is a transmembrane protein with an apparent relative molecular mass of 303 kDa. The cytoplasmic domain shows significant sequence identity to that of vertebrate classic cadherins. However, the extracellular domain is distinguished from its vertebrate counterparts by both an increased number of cadherin-specific repeats and the presence of four EGF-like repeats proximal to the transmembrane domain. Taken together, these data are consistent with the hypothesis that the sea urchin possesses several cadherins, including a novel member of the cadherin family, and that the dynamic regulation of cadherin localization plays a role in epithelial to mesenchymal conversions during gastrulation.

Gastrulation in the sea urchin involves an extensive rearrangement of cells of the archenteron giving rise to secondary mesenchyme at the archenteron tip followed by the foregut, midgut and hindgut. To examine the regulative capacity of this structure, pieces of the archenteron were removed or transplanted at different stages of gastrulation. After removal of any or all parts of the archenteron, the remaining veg 1 and/or veg 2 tissue regulated to replace the missing parts. Endoderm transplanted to ectopic positions also regulated to that new position in the archenteron. This ability to replace or regulate endoderm did not decline until after full elongation of the archenteron was completed. When replacement occurred, the new gut was smaller relative to the remaining embryo but the recognizable morphology of the archenteron was re-established. Long after the archenteron reveals territorial specification through expression of specific markers, the endodermal cells remain capable of being respecified to other gut regions. Thus, for much of gastrulation, the gut is conditionally specified. We propose that this regulative ability requires extensive and continuous short-range communication between cells of the archenteron in order to reorganize the tissues and position the boundaries of this structure even after experimental alterations.

The calcareous larval skeleton of euechinoid sea urchins is synthesized by primary mesenchyme cells which ingress prior to gastrulation. In embryos of the cidaroid sea urchin Eucidaris tribuloides, no mesenchyme cells ingress before gastrulation, yet larvae later contain skeletons. This apparent paradox is resolved by immunochemical, cell lineage and morphological evidence showing that spicule-forming cells of Eucidaris are homologous to primary mesenchyme cells of euechinoids. In particular, these two cell types share expression of two cell lineage-specific gene products, are derived from the same cellular precursors, the micromeres, and undergo a similar migratory phase prior to skeletogenesis. Despite these similarities, there are far fewer spicule-forming cells in Eucidaris than in typical euechinoids and they assume a different pattern during spiculogenesis. The homology between Eucidaris spicule-forming cells and euechinoid primary mesenchyme cells indicates that a heterochrony in the time of spicule-forming cell ingression has occurred since the divergence of their respective lineages.

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.

During sea urchin development, a tier-to-tier progression of cell signaling events is thought to segregate the early blastomeres to five different cell lineages by the 60-cell stage (E. H. Davidson, 1989, Development 105, 421-445). For example, the sixth equatorial cleavage produces two tiers of sister cells called ‘veg1’ and ‘veg2,’ which were projected by early studies to be allocated to the ectoderm and endoderm, respectively. Recent in vitro studies have proposed that the segregation of veg1 and veg2 cells to distinct fates involves signaling between the veg1 and veg2 tiers (O. Khaner and F. Wilt, 1991, Development112, 881-890). However, fate-mapping studies on 60-cell stage embryos have not been performed with modern lineage tracers, and cell interactions between veg1 and veg2 cells have not been shown in vivo. Therefore, as an initial step towards examining how archenteron precursors are specified, a clonal analysis of veg1 and veg2 cells was performed using the lipophilic dye, DiI(C16), in the seaurchin species, Lytechinus variegatus. Both veg1 and veg2 descendants form archenteron tissues, revealing that the ectoderm and endoderm are not segregated at the sixth cleavage. Also, this division does not demarcate cell type boundaries within the endoderm, because both veg1 and veg2 descendants make an overlapping range of endodermal cell types. The allocation of veg1 cells to ectoderm and endoderm during cleavage is variable, as revealed by both the failure of veg1 descendants labeled at the eighth equatorial division to segregate predictably to either tissue and the large differences in the numbers of veg1 descendants that contribute to the ectoderm. Furthermore, DiI-labeled mesomeres of 32-cell stage embryos also contribute to the endoderm at a low frequency. These results show that the prospective archenteron is produced by a larger population of cleavage-stage blastomeres than believed previously. The segregation of veg1 cells to the ectoderm and endoderm occurs relatively late during development and is unpredictable, indicating that later cell position is more important than the early cleavage pattern in determining ectodermal and archenteron cell fates.

We have investigated the distribution and function of an ezrin-radixin-moesin-like (ERM) molecule in the sea urchin. A sea urchin homologue of moesin was cloned that shares 75% amino acid similarity in the conserved N-terminal region to other moesin molecules. A 6.3 kb message is transcribed late in embryogenesis and is present in adult tissues. Polyclonal antibodies were generated to proteins expressed by a bacterial expression vector, and affinity purified. These antibodies recognize a single 75 kDa protein that is present throughout development in approximately equal abundance, and specifically they immuno-precipitate a single protein. We show by immunolocalization that SUmoesin has two predominant patterns during development. First, SUmoesin is rapidly redistributed after fertilization from a location throughout the egg cytoplasm to a location in the egg cortex. Later in embryogenesis, SUmoesin is localized to the apical ends of cells in the regions of cell-cell junctions. We show that SUmoesin is present in actin-rich regions of the embryo. Finally, we show that the location of SUmoesin requires an intact actin-based cytoskeleton. SUmoesin fails to localize to the plasma membrane after fertilization in the presence of cytocha-lasin B. Furthermore, SUmoesin loses its apical position in the region of cell-cell junctions in the presence of cytocha-lasin B in later stages of embryogenesis. This effect is reversible, and the microtubule inhibitor colchicine has no effect. These results show that SUmoesin becomes associated with apical plasma membrane structures early in development, and that SUmoesin is both coincident with actin and requires the assembly of actin filmaments to maintain its localization.

The sea urchin embryo follows a relatively simple cell behavioral sequence in its gastrulation movements. To form the mesoderm, primary mesenchyme cells ingress from the vegetal plate and then migrate along the basal lamina lining the blastocoel. The presumptive secondary mesenchyme and endoderm then invaginate from the vegetal pole of the embryo. The archenteron elongates and extends across the blastocoel until the tip of the archenteron touches and attaches to the opposite side of the blastocoel. Secondary mesenchyme cells, originally at the tip of the archenteron, differentiate to form a variety of structures including coelomic pouches, esophageal muscles, pigment cells and other cell types. After migration of the secondary mesenchyme cells from their original position at the tip of the archenteron, the endoderm fuses with an invagination of the ventral ectoderm (the stomodaem), to form the mouth and complete the process of gastrulation. A larval skeleton is made by primary mesenchyme cells during the time of archenteron and mouth formation. A number of experiments have established that these morphogenetic movements involve a number of cell autonomous behaviors plus a series of cell interactions that provide spatial, temporal and scalar information to cells of the mesoderm and endoderm. The cell autonomous behaviors can be demonstrated by the ability of micromeres or endoderm to perform their morphogenetic functions if either is isolated and grown in culture. The requirement for cell interactions has been demonstrated by manipulative experiments where it has been shown that axial information, temporal information, spatial information and scalar information is obtained by mesoderm and endoderm from other embryonic cells. This information governs the cell autonomous behavior and places the cells in the correct embryonic context

Unlabeled sponge aggregates were placed into suspensions of radioactively labeled sponge cells. All combinations of five species (Haliclona variabilis, Haliclona viridis, Tedania ignis, Homaxinella rudis,and Dysidea crawshayi) were used for aggregate collection experiments designed to test for species specificity of adhesion. Preliminary experiments had shown that freshly disaggregated cells from any two of the species would co-mingle during early aggregation. The aggregate collection system, however, showed time presence of adhesive specificity for all five species. Labeled and unlabeled cells became mixed when an unlabeled aggregate collected radioactive homotypic cells. Very few labeled cells were collected and mixing was not observed in heterotypic combinations.

OBSERVATIONS of developmental events such as the sorting out of cells during organogenesis suggest that highly specific mechanisms exist for cells to migrate apart or aggregate together. Several assays of cell-to-cell binding using cells from developing tissues have provided probable in vitro analogues of these recognition mechanisms. When cells from various organs of the embryo are dissociated, re-aggregation occurs in a tissue-specific manner with, for example, liver cells binding preferentially to other liver cells and brain cells binding preferentially to other brain cells. A number of groups using different assay systems have agreed with this general finding1–4. Furthermore, our own work with chick embryonic neural retina cells has shown that proteolytic enzyme treatment of these cells removes structures which are necessary for specific cell-cell adhesion to occur5. The relevant ‘recognition’ moieties on the cell surface may be glycoproteins6–8. We have investigated cell–cell adhesion using embryonic tissues from two strains of mice, C57B1/10 and C3H, which differ from one another at the H–2 locus as well as at a number of minor H–loci.

In order to study the biophysical forces involved in cell‐substrate (or cell‐cell) adhesion, it is necessary to measure the strength of adhesion. Two questions can be addressed using the centrifugal cell adhesion assay provided in this unit: what is the ligand‐receptor affinity for cells adhering at 4oC and what is the strength of the ATP‐dependent processes that strengthen adhesion at 37oC. In both cases, the strength of adhesion is measured as the resistance to disruption in the presence of a measured centrifugal force.