Serum effects on the in vitro differentiation of sea urchin micromeres

When micromeres isolated from the 16-cell stage of Strongylocentrotus purpuratus are cultured in sea water containing 3.5% horse serum, they produce spicules at approximately the same time as in normal development. The serum requirement of the micromeres has been investigated by adding serum at varying intervals after isolation or by pulsing the cells with serum at specific times during their in vitro development. The optimum time of serum addition for spicule formation is 36 h after fertilization (AF). Further delay in the addition of serum results in a reduction in the number of spicules formed in culture and a delay in the time at which they appear. A 1-h pulse of serum at 36 h AF is sufficient to initiate a response in some of the micromere aggregates. A 12-h pulse at 36 h AF produces the maximum number of spicules per culture. The critical period for serum addition, 36-48 h AF, corresponds to the time in the normal embryo at which the syncytial primary mesenchyme ring is formed. Electron micrographs of cultured cells demonstrate that micromeres cultured without serum until 48 h AF fail to form pseudopodial extensions and remain as rosette-like clusters of cells. If serum is present, extensive pseudopodial networks form which resemble the primary ring syncytium. These results suggest that serum acts to stimulate fused pseudopodial networks in cultures of micromeres and that the resulting syncytium is necessary for spicule formation.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

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.     

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.     

Matrix and Mineral in the Sea Urchin Larval Skeleton

The endoskeletal spicules of sea urchin larvae are composed of calcite, a surrounding extracellular matrix, and small amounts of occluded matrix proteins. The spicules are formed by primary mesenchyme cells (PMCs) in the blastocoel of the embryo, where they adopt stereotypical locations, thereby specifying where spicules will form. PMCs also fuse to form cytoplasmic cords connecting the cell bodies, and it is within the cords that spicules arise. The mineral phase contains 5% Mg as well as Ca, and about 0.1% of the mass is protein. The matrix and mineral form concentric plies, and the composite has different physical properties than those of pure calcite. The calcite diffracts as a single crystal and is composed of well-ordered, but not perfectly ordered, microdomains. There is evidence for adsorption of matrix proteins to specific crystal faces at domain boundaries, which may help regulate crystal growth and texture. Immature spicules contain considerable precipitated amorphous CaCO3, and PMCs also have vesicles that contain amorphous CaCO3. This suggests the hypothesis that the cellular precursor to the spicules is actually amorphous CaCO3 stabilized in the cell by protein. The spicule s enveloped by the PMC cord, but is topologically exterior to the cell. The PMC plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tip. The characteristics, localization, and possible function of the four identified matrix proteins are discussed. SM50, SM37, and PM27 all primarily enclose the mineral, though small amounts are occluded. SM30 is found in cellular vesicles and is probably the principal occluded protein of the spicule.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

Role of LSM34/SpSM50 proteins in endoskeletal spicule formation in sea urchin embryos

Abstract. Sea urchin embryos form an endoskeletal spicule composed of calcium carbonate and occluded matrix proteins. The accumulation of the LSM34 spicule matrix protein in embryos of Lytechinus pictus (and its ortholog, SpSM50, in Strongylocentrotus purpuratus ) has been inhibited using morpholino antisense oligonucleotides. The inhibition, using relatively high levels of antisense reagent, can result in the complete absence of spicules, and the complete loss of immunoreactive LSM34/SpSM50, as judged by immunostaining and Western blotting. Primary mesenchyme cells (PMCs) do form and express PMC-specific cell surface antigens despite this inhibition. However, these anti-LSM34/SpSM50-treated embryos do not accumulate SM30 protein, another major matrix protein. Hence, both the initiation of spicule formation and subsequent morphogenesis require LSM34 accumulation in L. pictus , and the accumulation of its ortholog, SpSM50, in S. purpuratus .     

The origin of skeleton forming cells in the sea urchin embryo

Summary In embryos of the modern sea urchin species, subclass Euechinoidea, primary mesenchyme cells are derived from the progeny of micromeres formed at the sixteen cell stage of embryogenesis. The micromeres reside within the vegetal plate epithelium and later ingress into the blastocoel as primary mesenchyme cells which form the larval skeleton. Embryos of Eucidaris tribuloides, a member of the primitive subclass Perischoechinoidea, exhibit several noteworthy differences from euechinoid primary mesenchyme cell lineage including variable numbers and sizes of micromeres, the absence of mesenchyme ingression, and the lack of any detectable primary mesenchyme although a larval skeleton forms. In the present study, the cell lineage of the spiculogenic mesenchyme has been studied in Eucidaris tribuloides and in the euechinoid Lytechinus pictus by microinjecting the fluorescent tracer, Lucifer Yellow, into individual blastomeres of the embryo. In addition, wheat germ agglutinin, a lectin which binds only to primary mesenchyme cells of the early euechinoid embryo, was injected into the blastocoel of embryos of both species in order to examine the distribution of cells which possess primary mesenchyme-specific cell surface markers. The results of these experiments demonstrate that the spiculogenic mesenchyme of both Lytechinus and Eucidaris arise from descendants of micromeres formed at the sixteen cell stage, although the temporal and spatial distribution of these mesenchyme cells varies considerably between species. Furthermore, the evidence obtained suggests that the information necessary for spicule formation is already segregated to the vegetal pole by the eight cell stage. The results also suggest that there are no gap junctions present between the blastomeres of the early sea urchin embryo.     

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A--gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

The organic matrix of the skeletal spicule of sea urchin embryos

The micromeres that arise at the fourth cell division in developing sea urchin embryos give rise to primary mesenchyme, which in turn differentiates and produces calcareous endoskeletal spicules. These spicules have been isolated and purified from pluteus larvae by washing in combinations of ionic and nonionic detergents followed by brief exposure to sodium hypochlorite. The spicules may be demineralized and the integral matrix dissolves. The matrix is composed of a limited number of glycoproteins rich in aspx, glux, gly, ser, and ala, a composition not unlike that found in matrix proteins of biomineralized tissues of molluscs, sponges, and arthropods. There is no evidence for collagen as a component of the matrix. The matrix contains N-linked glycoproteins of the complex type. The matrix arises primarily from proteins synthesized from late gastrulation onward, during the time that spicule deposition occurs. The mixture of proteins binds calcium and is an effective immunogen. Electrophoresis of the glycoproteins on SDS-containing acrylamide gels, followed by blotting and immunocytochemical detection, reveals major components of approximately 47, 50, 57, and 64 kD, and several minor components. These same components may be detected with silver staining or fluorography of amino acid-labeled proteins. In addition to providing convenient molecular marker for the study of the development of the micromere lineage, the spicule matrix glycoproteins provide an interesting system for investigations in biomineralization.     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Cellular control over spicule formation in sea urchin embryos: A structural approach

The spicules of the sea urchin embryo form in intracellular membrane-delineated compartments. Each spicule is composed of a single crystal of calcite and amorphous calcium carbonate. The latter transforms with time into calcite by overgrowth of the preexisting crystal. Relationships between the membrane surrounding the spiculogenic compartment and the spicule mineral phase were studied in the transmission electron microscope (TEM) using freeze-fracture. In all the replicas observed the spicules were tightly surrounded by the membrane. Furthermore, a variety of structures that are related to the material exchange process across the membrane were observed. The spiculogenic cells were separated from other cell types of the embryo, frozen, and freeze-dried on the TEM grids. The contents of electron-dense granules in the spiculogenic cells were shown by electron diffraction to be composed of amorphous calcium carbonate. These observations are consistent with the notion that the amorphous calcium carbonate-containing granules contain the precursor mineral phase for spicule formation and that the membrane surrounding the forming spicule is involved both in transport of material and in controlling spicule mineralization.     

The role of micromere signaling in Notch activation and mesoderm specification during sea urchin embryogenesis.

In the sea urchin embryo, the micromeres act as a vegetal signaling center. These cells have been shown to induce endoderm; however, their role in mesoderm development has been less clear. We demonstrate that the micromeres play an important role in the induction of secondary mesenchyme cells (SMCs), possibly by activating the Notch signaling pathway. After removing the micromeres, we observed a significant delay in the formation of all mesodermal cell types examined. In addition, there was a marked reduction in the numbers of pigment cells, blastocoelar cells and cells expressing the SMC1 antigen, a marker for prospective SMCs. The development of skeletogenic cells and muscle cells, however, was not severely affected. Transplantation of micromeres to animal cells resulted in the induction of SMC1-positive cells, pigment cells, blastocoelar cells and muscle cells. The numbers of these cell types were less than those found in sham transplantation control embryos, suggesting that animal cells are less responsive to the micromere-derived signal than vegetal cells. Previous studies have demonstrated a role for Notch signaling in the development of SMCs. We show that the micromere-derived signal is necessary for the downregulation of the Notch protein, which is correlated with its activation, in prospective SMCs. We propose that the micromeres induce adjacent cells to form SMCs, possibly by presenting a ligand for the Notch receptor.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.