Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.     

Signals from primary mesenchyme cells regulate endoderm differentiation in the sea urchin embryo

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm-specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.     

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.     

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.     

Proto-oncogene homologous sequences in the sea urchin genome

Using probes specific for several oncogenes/proto-oncogenes we have performed gel blot hybridization analyses of genomic DNA isolated from the sea urchinStrongylocentrotus droebachiensis. Probes prepared from v-erbB, v-myc, c-myb and v-fps were found to hybridize with discrete fragments of HindIII digested genomic DNA. In contrast, probes prepared from v-abl, v-fos, v-sis, v-src, and v-mos either hybridized with multiple fragments, indicating non-specific binding, or failed to hybridize at all above background levels. These results clearly demonstrate the presence of proto-oncogene homologous sequences in the sea urchin genome.     

Outgrowth of pseudopodial cables induced by all-trans retinoic acid in micromere-derived cells isolated from sea urchin embryos

Cultured cells derived from micromeres of sea urchin embryos underwent pseudopodial cable growth without spicule rod formation in the presence of all-trans retinoic acid (tRA) or insulin. Pseudopodial cable growth caused by tRA or insulin was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation of protein tyrosine residue was augmented in the cells treated with tRA or insulin and was inhibited by genistein. Probably, protein tyrosine kinase takes an indispensable part in signal transduction systems for tRA and insulin in these cells. In tRA-treated cells, augmentation of the phosphorylation of protein tyrosine residue was accompanied by an increase in the activity of protein tyrosine kinase and was inhibited by actinomycin D, inhibiting cable growth. Activation of this enzyme in tRA-treated cells probably depends on RNA synthesis. In insulin-treated cells, augmentation of tyrosine residue phosphorylation occurred without any appreciable change in this enzyme's activity and was hardly affected by actinomycin D. Phosphorylation of protein tyrosine residue seems to be activated by the binding of insulin to an insulin receptor. Pseudopodial cable growth in these cells treated with tRA or insulin was inhibited by wortmannin. Phosphatidylinositol 3 kinase probably participates in tRA and insulin signal transduction systems.     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

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.     

Specification and differentiation processes of secondary mesenchyme-derived cells in embryos of the sea urchin Hemicentrotus pulcherrimus

Four types of mesoderm cells (pigment cells, blastocoelar cells, coelomic pouch cells and circumesophageal muscle cells) are derived from secondary mesenchyme cells (SMC) in sea urchin embryos. To gain information on the specification and differentiation processes of SMC-derived cells, we studied the exact number and division cycles of each type of cell in Hemicentrotus pulcherrimus. Numbers of blastocoelar cells, coelomic pouch cells and circumesophageal muscle fibers were 18.0 +/- 2.0 (36 h post-fertilization (h.p.f.)), 23.0 +/- 2.5 (36 h.p.f.) and 9.5 +/- 1.3 (60 h.p.f.), respectively, whereas the number of pigment cells ranged from 40 to 60. From the diameters of blastocoelar cells and coelomic pouch cells, the numbers of division cycles were elucidated; these two types of cells had undertaken 11 rounds of cell division by the prism stage, somewhat earlier than pigment cells. To determine the relationship among the four types of cells, we tried to alter the number of pigment cells with chemical treatment and found that CH3COONa increased pigment cells without affecting embryo morphology. Interestingly, the number of blastocoelar cells became smaller in CH3COONa-treated embryos. In contrast, blastocoelar cells were markedly increased with NiCl2 treatment, whereas the number of pigment cells was markedly decreased. The number of coelomic pouch cells and circumesophageal muscle fibers was not affected with these treatments, indicating that coelomic pouch and muscle cells are specified independently of, or at much later stages, than pigment and blastocoelar cells.     

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

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

Genes of the sea urchin embryo: An annotated list as of December 1994

The main literature regarding gene structure and expression in sea urchin embryos is schematically reported and briefly commented upon. Although the subject has expanded particularly over the last 10 years, to which the review mostly refers, some historical reference is also given. More space is reserved to the regulation of the synthesis of histones and cytoskeletal actins, where the attention of various authors has been especially present; the regulation of such a synthesis is described both at a territorial level and a temporal level during the sea urchin development.     

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.     

Involvement of Delta and Nodal signals in the specification process of five types of secondary mesenchyme cells in embryo of the sea urchin, Hemicentrotus pulcherrimus

Secondary mesenchyme cells (SMCs) of the sea urchin embryo are composed of pigment cells, blastocoelar cells, spicule tip cells, coelomic pouch cells and muscle cells. To learn how and when these five types of SMCs are specified in the veg₂ descendants, Notch or Nodal signaling was blocked with γ‐secretase inhibitor or Nodal receptor inhibitor, respectively. All types of SMCs were decreased with DAPT, while sensitivity to this inhibitor varied among them. Pulse‐treatment revealed that five types of SMCs are divided into “early” (pigment cells and blastocoelar cells) and “late” (spicule tip cells, coelomic pouch cells and muscle cells) groups; the “early” group was sensitive to DAPT up to the hatching, and the “late” group was sensitive until the mesenchyme blastula stage. Judging from timing of the shift of Delta‐expressing regions, it was suggested that the “early” group and “late” groups are derived from the lower and the middle tier of veg₂ descendants, respectively. Interestingly, numbers of SMCs were also altered with SB431542; blastocoelar cells, coelomic pouch cells and circum‐esophageal muscles decreased, whereas pigment cells and spicule tip cells increased in number. Pulse‐treatment showed that the “early” group was sensitive up to the mesenchyme blastula stage, while the “late” group up to the onset of gastrulation. Thus, it became clear that precursor cells of the “early” and “late” groups, which are located in different regions in the vegetal plate, receive Delta and Nodal signals at different timings, resulting in the diversification of SMCs. Based on the obtained results, the specification processes of five types of SMCs are diagrammatically presented.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

Protein synthetic activity in swimming sea urchin spermatozoa

Summary

Free swimming Paracentrotus lividus spermatozoa show a significant rate of protein synthesis which remains nearly linear over a period of 90 min. This protein synthetic activity is scarcely affected by emetine but completely suppressed by chloramphenicol, suggesting its possible mitochondrial origin.     

Relationship between ATP level and respiratory rate in sea urchin embryos

In sea urchin embryos, the rate of respiration, as a result of electron transport through the mitochondrial respiratory chain, was enhanced after hatching without any change in the intrinsic capacity of electron transport in mitochondria. The increase in respiratory rate after hatching was accompanied by an evident decrease in intracellular adenosine triphosphate (ATP) concentration without any change in intracellular levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Adenosine triphosphate is proposed to fortify acceptor control of respiration at high concentrations and to reduce the respiratory rate even in the presence of ADP, the acceptor. The relationships between the respiratory rate and intracellular ATP concentration in embryos were the same as those in mitochondria isolated from embryos, obtained in the presence of ADP at the same concentration as in the embryos. Probably, the respiratory rate is enhanced after hatching because of the decrease in the level of ATP. In embryos kept in a medium containing adenosine, intracellular ATP concentration increased especially after hatching, without any change in the ADP level, and the respiratory rate after hatching was made as low as the rate expected, based on the relationships obtained on isolated mitochondria. The respiratory rate in embryos probably depends on intracellular ATP concentration, irrespective of the developmental stage in early development.