The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms

Adult tissues of the sea urchin, Strongylocentrotus purpuratus, were analyzed for the products of a set of genes whose expression, in the embryo, is restricted to the skeletogenic primary mesenchyme (PM). Three embryonic PM-specific mRNAs were found to be abundant in adult skeletal tissues (test and lantern), but not in a variety of soft tissues. Homologous mRNAs were also found in skeletal tissues of the congeneric sea urchin, S. droebachiensis, as well as a more distantly related echinoid, Dendraster excentricus, and an asteroid, Evasterias troschellii. The distributions of two of these RNAs were analyzed in regenerating spines of adult S. purpuratus using in situ hybridization. These gene products were localized primarily in the calcoblasts that accumulated at the regeneration site. In nonregenerating spines SpLM 18 RNAs, the most abundant of these gene products, were localized in a small population of noncalcoblast cells scattered through the spine shaft, and were absent from calcoblasts. These observations suggest that a program of gene expression associated with the process of calcification is conserved both developmentally through the period of metamorphosis and evolutionarily among the echinoderms.     

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.     

Unequal divisions at the third cleavage increase the number of primary mesenchyme cells in sea urchin embryos

To clarify the distribution and behavior of the maternal factors that direct the differentiation of primary mesenchyme cells (PMC) in sea urchin embryos, unequal division was induced at the third cleavage with the treatment of dinitro-phenol (DNP), and the numbers of differentiated PMC were examined. The most surprising finding was that the number of PMC was considerably increased in some of the DNP-treated embryos. This increase in the number of PMC was suggested to be closely related to the size of the precocious micromeres formed at the 8-cell stage. By measuring both the size of the precocious micromeres and the number of PMC in individual embryos, it was suggested that almost all the descendants of the precocious micromeres differentiated into PMC, if the volume was less than 26 pL (about three times the volume of normal micromeres). Cell tracing experiments ascertained that precocious micromeres with small volumes behave just like micromeres formed at the fourth cleavage in normal embryos. The obtained results indicated that the maternal factors present in sea urchin embryos can direct, at least, more than three times the number of PMC, and that the number of cell divisions of the PMC lineage is not strictly regulated.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

Synthesis of RNA by dissociated cells of the sea urchin embryo

The incorporation of radioactive uridine into RNA by micromeres, mesomeres and macromeres of sea urchin embryos was studied, employing methods for separating the cell types in pure suspension. At the 16-cell stage, the 3-cell types, on a per genome basis, synthesized RNA at approximately the same rate although on a per mg protein basis the micromere-RNA synthetic rate was considerably higher than either mesomeres or macromeres. At the 32-cell stage, incorporation of radioactive uridine by micromeres decreased relative to mesomeres and macromeres. It was demonstrated that radioactive uridine could not be effectively washed or diluted out of the cells of 16-cell stage embryos. Experiments on reaggregating cells did not detect any transfer or transport of radioactivity from micromeres to the other cells. Possible explanations for these findings versus the disparate results of previous investigators were presented.     

THE SYNTHESIS OF 5S RNA AND ITS REGULATION DURING EARLY SEA URCHIN DEVELOPMENT

De novo synthesis of 5S RNA and of transfer RNA (tRNA) has been demonstrated previously to occur by mid-cleavage (128-cell stage) in sea urchin embryos (24). The present study focused on determining more precisely the time of onset of activity of the genes for 5S RNA and for tRNA during sea urchin embryogenesis by preloading the GTP precursor pools of unfertilized eggs. The results showed that newly-made 5S RNA and tRNA could be detected as early as the 32-cell stage. In order to determine whether newly-synthesized 5S RNA accumulates coordinately during development with newly-made 26S (34) and 18S ribosomal RNAs (rRNAs), the relative rates of accumulation of these three RNA molecules were measured and compared at each of several stages of sea urchin embryogenesis. In contrast to the coordinated accumulation of newly-synthesized 26S and 18S rRNAs, newly-made 5S RNA accumulated in excess at the mesenchyme blastula (9-fold excess), midgastrula (5-fold excess) and prism (3-fold excess) stages. The 5S RNA/26S RNA molar ratios only approached unity in advanced (48 hr) plutei. The non-coordinated accumulation of newly-made 5S RNA with that of 26S and 18S rRNAs suggests that the accumulation of these newly-synthesized RNAs is differentially regulated during early sea urchin development.     

Centrifugal elutriation of large fragile cells: isolation of RNA from fixed embryonic blastomeres.

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.     

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.     

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.     

Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.     

Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: Cytoplasmic retention of SpOtx possibly mediated through an α-actinin interaction

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-vegetal axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities. Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained In the cytoplasm at early developmental stages we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be α-actinin. A yeast two-hybrid analysis showed a specific interaction between the proline-rich region of SpOtx and a putative SH3 domain of the sea urchin α-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to α-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate. © 1996 Wiley-Liss, Inc.     

Changes in Insulin-Binding Capacity of the Plasma Membrane Fraction during Culture in vitro of Cells Derived from Micromeres of 16-Cell-Stage Sea Urchin Embryos

In cultured cells derived from isolated micromeres of 16-cell stage sea urchin embryos, which undergo insulin-induced pseudopodial cable growth, specific and reversible insulin binding by a 52-kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin-binding capacity in micromere-derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U-³H]uridine incorporation into RNA and [³⁵S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres. The capacity in the plasma membrane fraction increased, accompanied by its decrease in the microsome fraction, during 5 h of culture of micromere-derived cells. The insulin receptor is probably accumulated in microsomes of presumptive micromeres prior to the 16-cell stage and transferred to the plasma membrane, resulting in an increase in the insulin binding capacity of micromere-derived cells during 5 h of culture.     

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.