Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

Occurrence of fibronectin on the primary mesenchyme cell surface during migration in the sea urchin embryo

The distribution of fibronectin in situ in the sea urchin embryo was examined by using indirect immunofluorescence with an antibody raised against human plasma fibronectin. Fibronectin was detected on the surfaces of primary mesenchyme cells in the mid-mesenchyme blastula stage, when these cells are migratory. However, it was not detected on these cells at the early mesenchyme blastula or early gastrula stages. Also, it was not detected in the blastocoel nor on the basal surface of the blastular wall. The migration of the primary mesenchyme cells is therefore correlated with a stage-dependent occurrence of cell surface-associated fibronectin.     

Occurrence of Fibronectin on the Primary Mesenchyme Cell Surface During Migration in the Sea Urchin Embryo

The distribution of fibronectin in situ in the sea urchin embryo was examined by using indirect immunofluorescence with an antibody raised against human plasma fibronectin. Fibronectin was detected on the surfaces of primary mesenchyme cells in the mid-mesenchyme blastula stage, when these cells are migratory. However, it was not detected on these cells at the early mesenchyme blastula or early gastrula stages. Also, it was not detected in the blastocoel nor on the basal surface of the blastular wall. The migration of the primary mesenchyme cells is therefore correlated with a stage-dependent occurrence of cell surface-associated fibronectin.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

Behavior of Primary Mesenchyme Cells In situ Associated with Ultrastructural Alteration of the Blastocoelic Material in the Sea Urchin, Anthocidaris crassispina

In the blastula of the sea urchin, Anthocidaris crassispina , a small number of primary mesenchyme cells (PMCs) ingressed from the blastocoel wall taking a bottle shape. The majority of the PMCs followed the first group of PMCs. These ingressed without taking the bottle shape, and became round within the blastocoel wall. After ingression, the PMCs migrated as single cells retaining their round cell contour. The average velocity of their migration was 13.3 μm/hr.
The blastocoel contained Alcian blue (pH 1.0)-positive material which changed its light microscopic configuration from being amorphous in the hatched and mesenchyme blastulae to being fibrous in the early gastrulae. Ultrastructurally, the blastocoelic material in the hatched blastulae was composed of 27 nm diameter granules. In the mesenchyme blastulae and the early gastrulae relatively long 15 nm diameter fibers were seen in addition to the 27 nm diameter granules. The 27 nm diameter granules bound the ruthenium red while the 15 nm diameter fibers did not. The 27 nm diameter granules formed aggregates in the hatched blastulae, and were bound to the 15 nm diameter fibers in the mesenchyme blastulae and early gastrulae to form a fibrous network which was observed by a light microscope.     

Studies on the cellular basis of morphogenesis in the sea urchin embryo. Directed movements of primary mesenchyme cells in normal and vegetalized larvae

A time-lapse study has been made of the movements of the primary mesenchyme cells in the developing sea urchin larva. It shows that these cells move by pseudopod formation and contraction, and that a transition takes place--within a few hours--from a more or less random cluster, in the early mesenchyme blastula, to a well-organized, coherent pattern on the ectoderm of the gastrula. This organization is achieved by a striking random exploration of the wall of the larva by the pseudopods, followed by their contraction. The final pattern of the mesenchyme reflects those regions of the wall where the contacts between pseudopods and wall are most stable. The mechanism is thus one of selective fixation rather than of selective conduction. The pseudopodal contacts are seen to be continually made and broken, even when the final pattern is formed. The pseudopods of several cells may fuse to form a common pseudopod, these cells then migrating together. This is particularly evident in vegetalized larvae, but is also typical of the ventral side. Despite considerable variations in the way in which the final pattern is achieved, several main phases can be distinguished. The first is a radial displacement of the cells from the vegetal plate onto the presumptive ectoderm, followed by a phase of dispersion. The cells then gradually accumulate at a characteristic level, and form a ring. During this process, and when the ring is formed, the cells tend to accumulate in two clusters along the ring. The pseudopods of the cells in these clusters join into a cable, the end of which is highly branched; it explores the ectoderm, and extends the cell clusters to form branches from the ring. In vegetalized larvae, the pattern of distribution is simplified, but the same principles apply. It is suggested that the variations in the way in which the pattern is achieved are, in all probability, merely a reflexion of the lack of precision in the time sequence of changes in adhesive properties of the primary mesenchyme and blastocoel wall.     

The insertion of mesenchyme cells into the ectoderm during differentiation in Sea urchin embryos

Summary During the course of sea urchin development, from early blastula to pluteus larva, there are two major visible processes toward which all activities seem to be focused. They are the differentiation of the larval skeleton by the primary mesenchyme cells and the differentiation of the primitive gut by the secondary mesenchyme cells. These activities take place within the shell-like layer of epithelial cells, or ectodermal wall. The interactive role of the ectodermal wall with the mesenchyme cells is not yet clearly understood. A number of earlier studies have proposed that the ectoderm may have an inductive influence on the mesenchyme cells and that its inner surface forms a molecular template for guiding the mesenchyme cells. In this report, we suggest an additional role for the ectodermal wall. We show that some primary mesenchyme cells and secondary mesenchyme cells insert between the cells of the ectodermal wall in order to firmly anchor the anlage of the larval skeleton and primitive gut during differentiation. This mechanism may provide a physical basis for maintaining the stable positional relationship of the anlage during development.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

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.     

Sea urchin primary mesenchyme cells: relation of cell polarity to the epithelial-mesenchymal transformation

In euechinoid sea urchin embryos, a subset of epithelial cells in the wall of the blastula become pulsatile, elongate, lose connections with their neighboring cells, and move into the blastocoel to form the primary mesenchyme cells. The Golgi apparatus and microtubule organizing center (MTOC) are located at the apical end of these epithelial cells. We show that as primary mesenchyme cells begin to move into the blastocoel, the Golgi apparatus and MTOC move to a new position adjacent to the apical side of the nucleus. They do not move to a position between the nucleus and the leading (i.e., basal) end of the cell as they do in cultured fibroblasts undergoing directed migration. In addition, we have inhibited the movement of membranous vesicles to the cell surface by incubating embryos in the ionophore monensin. We have used antibodies to msp130, a primary mesenchyme cell surface-specific glycoprotein, to demonstrate that monensin inhibits the movement of msp130-containing vesicles to the cell surface. Despite the inhibition of membrane shuttling by monensin, primary mesenchyme cells ingress on schedule and display normal cell-shape changes. We draw two conclusions from our data. First, the cellular elongation that characterizes ingression is not due to the local insertion of membrane at the leading (basal) end of the cell. Second, ingression does not depend upon establishment of the same cell polarity required for fibroblasts to carry out directed cell migration.     

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.     

Placentation in the degu (Octodon degus): Analogies with extrasubplacental trophoblast and human extravillous trophoblast

This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na⁺/K⁺ ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na⁺/K⁺ ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.     

Primary mesenchyme cell patterning during the early stages following ingression

Sea urchin primary mesenchyme cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-cell stage, and later the 2 unlabeled micromeres are removed at the 16-cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-cell stage shows a 2-step pattern of localization. At late mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.     

Isolation,culture, and differentiation of echinoid primary mesenchyme cells

Summary Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.     

Microfilaments, cell shape changes, and the formation of primary mesenchyme in sea urchin embryos.

Primary mesenchyme formation in sea urchin embryos occurs when a subset of epithelial cells of the blastula move from the epithelial layer into the blastocoel. The role of microfilaments in producing the cell shape changes that characterize this process, referred to as ingression, was investigated in this study. f-Actin was localized by confocal microscopy using labeled phalloidin. The distribution of f-actin was observed before, during, and after ingression and was correlated with cellular movements. Prior to the onset of ingression, staining became intense in the apical region of putative primary mesenchyme and disappeared following the completion of mesenchyme formation. The apical end of these cells constricted coincidentally with the appearance of the intensified staining, indicating that f-actin may be involved in this constriction. In addition, papaverine, a smooth muscle cell relaxant that interferes with microfilament-based contraction, and that was shown in this study to inhibit cytokinesis, diminished apical constriction and delayed ingression. Despite this interference with apical constriction, the basal surface of ingressing cells protruded into the blastocoel. It is suggested that apical constriction, while not necessary for ingression, does contribute to the efficient production of mesenchyme and that protrusion of the basal surface results from changes that occur independent of apical constriction.     

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.