Electron microscopic studies on primary mesenchyme cell ingression and gastrulation in relation to vegetal pole cell behavior in sea urchin embryos

Early morphogenetic events of primary mesenchyme cell (PMC) ingression and gastrulation were examined by scanning and transmission electron microscopy, with special attention directed to changes in the shape of vegetal pole cells, the length of their microvilli, and interactions between microvilli and the hyaline layer (HL). Eight cells (vegetal pole cells) with elongated microvilli remained in the vegetal pole region while surrounding cells ingressed into the blastocoel to form the primary mesenchyme. These vegetal pole cells indented with the surrounding cells at the stage of gastrulation. The outer surface area with elongated microvilli of vegetal pole cells expanded at the stage of PMC ingression, but was considerably reduced at gastrulation. Microvilli on vegetal pole cells continued to adhere to the HL up to the stage of PMC ingression, but ceased to do so at the time of gastrulation. Thus, the area with separated HL, which is restricted to the region of the PMC released at the stage of PMC ingression, spreads almost entirely throughout the area of the indenting vegetal plate at gastrulation. The apical lamina, apparently consisting of fibrous material intertwinning the stalks of the microvilli, filled the space between the HL and ectodermal cells. The cells surrounding those of the vegetal pole and indenting with those at the stage of gastrulation appeared to behave in the same way as ingressing PMCs in both cell-shape and loss of adhesion of microvilli to HL. The role of vegetal pole cells in early morphogenetic events is discussed.     

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.     

The origin of spicule-forming cells in a 'primitive' sea urchin (Eucidaris tribuloides) which appears to lack primary mesenchyme cells

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

Evolutionary modification of mesenchyme cells in sand dollars in the transition from indirect to direct development

Peronella japonica, an intermediate type of direct-developing sand dollar, forms an abbreviated pluteus, followed by metamorphosis within 3 days without feeding. In this species, ingression of mesenchyme cells starts before hatching and continues until gastrulation, but no typical secondary mesenchyme cells (SMCs) migrate from the tip of the archenteron. Here, I investigated the cell lineage of mesenchyme cells through metamorphosis in P. japonica and found that mesenchyme cells migrating before hatching (early mesenchyme cells [EMCs]) were exclusively derived from micromeres and became larval skeletogenic cells, whereas cells migrating after hatching (late mesenchyme cells [LMCs]) appeared to contain several nonskeletogenic cells. Thus, it is likely that EMCs are homologous to primary mesenchyme cells (PMCs) and LMCs are similar to the SMCs of typical indirect developers, suggesting that heterochrony in the timing of mesenchyme cell ingression may have occurred in this species. EMCs disappeared after metamorphosis and LMCs were involved in adult skeletogenesis. Embryos from which micromeres were removed at the 16-cell stage formed armless plutei that went on to form adult skeletons and resulted in juveniles with normal morphology. These results suggest that in P. japonica, LMCs form adult skeletal elements, whereas EMCs are specialized for larval spicule formation. The occurrence of evolutionary modifications in mesenchyme cells in the transition from indirect to direct development of sand dollars is discussed.     

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2∷GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.     

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.     

The cleavage initiation site establishes the posterior pole of the hydrozoan embryo

Summary When the first cleavage of the hydrozoan egg is reversibly suppressed, two cleavage furrows frequently form simultaneously at the time of the second cleavage. If these two cleavage initiation sites are far enough apart, each one specifies a site of gastrulation, and the embryo that forms develops into a two tailed planula larva. When two tailed planulae are induced to metamorphose, they form a polyp with two stalks and hydranths.     

Gastrulation in Halocordyle disticha (Hydrozoa,Athecata)

Summary

Previous reports of development in Halocordyle disticha have described gastrulation as occurring by gradual differentiation of the inner and outer cells of the stereoblastula. In 1984, however, Martin and Thomas described an indentation on the surface of the embryo at the time of gastrulation. They hypothesized from morphological data that the indentation represented a blastopore. Here we provide results of marking studies which demonstrate that the indentation is in fact a site of cellular ingression. This is the first example known of gastrulation that involves unipolar ingression in a form with a stereoblastula. Possible functions of gastrulation by unipolar ingression are discussed, and the possible phylogenetic significance of the occurrence of such a mode of gastrulation in H. disticha is considered.     

Timing system for the start of gastrulation in the Xenopus embryo

This study examined which component of the egg, the nucleus or cytoplasm, is involved in the timing of the start of gastrulation in the Xenopus embryo, and when it starts to measure time. First, nuclei of cells of 256-cell stage embryos were transplanted to enucleated eggs 60 min after activation. These eggs showed first cleavage 20-30 min later than control eggs fertilized at the same time as the activation of recipient eggs, and started gastrulation 25-35 min later than control embryos (depending on the delay in the first cleavage). Second, eggs whose nuclei were temporarily isolated by the extrusion of the portion containing the nucleus out of the fertilization envelope showed first cleavage 60-90 min later than sibling control eggs, because of delayed introduction of the nucleus from the extruded portion. They started gastrulation 60-90 min later than sibling control embryos (depending on the delay in the first cleavage). The portion inside the envelope underwent two to three rounds of oscillation in cell cycle relevant activities before the first cleavage, while the portion outside underwent the same rounds of cleavage as the inside portion. From the present and previous results it is concluded that the putative timing system for the start of gastrulation in the Xenopus embryo, whether it consists of a single or of multiple clocks, starts measuring time at or around the first cleavage, and that the presence of both the nucleus and the cytoplasm in the same cell and occurrence of mitosis and/or cleavage there are indispensable for the timing system to work, although the role of the cytoplasm is superior to that of the nucleus.     

Embryonic cleavage cycles: how is a mouse like a fly?

The evolutionary advent of uterine support of embryonic growth in mammals is relatively recent. Nonetheless, striking differences in the earliest steps of embryogenesis make it difficult to draw parallels even with other chordates. We suggest that use of fertilization as a reference point misaligns the earliest stages and masks parallels that are evident when development is aligned at conserved stages surrounding gastrulation. In externally deposited eggs from representatives of all the major phyla, gastrulation is preceded by specialized extremely rapid cleavage cell cycles. Mammals also exhibit remarkably fast cell cycles in close association with gastrulation, but instead of beginning development with these rapid cycles, the mammalian egg first devotes itself to the production of extraembryonic structures. Previous attempts to identify common features of cleavage cycles focused on post-fertilization divisions of the mammalian egg. We propose that comparison to the rapid peri-gastrulation cycles is more appropriate and suggest that these cycles are related by evolutionary descent to the early cleavage stages of embryos such as those of frog and fly. The deferral of events in mammalian embryogenesis might be due to an evolutionary shift in the timing of fertilization.     

Changes in voltage-dependent ion currents during meiosis and first mitosis in eggs of an ascidian.

Different patterns of voltage-dependent ion currents are present in mature eggs and in early embryos of the ascidian Boltenia villosa, as if each ion current is regulated in a different manner between fertilization and the early cleavages of embryogenesis. The ion currents appear and/or disappear with precise timing suggesting that they play important roles at specific times during early development. We investigated changes in three voltage-dependent ion currents (an inwardly rectifying chloride current, a calcium current, and a sodium current) and membrane surface area over time between the resumption of meiosis (with fertilization or activation) and the first mitotic cleavage. Using time-lapse video recordings made during whole-cell patch-clamp experiments, we were able to correlate electrophysiological changes with morphological changes and cell cycle related events. Between fertilization and first cleavage, INa was lost exponentially, the density of ICa remained relatively constant, and the amplitudes of both ICl and membrane surface area fluctuated in time with the cell cycle. ICl and surface area increased whenever the cell began dividing--with the polar body extrusions and the formation of the first cleavage furrow. This suggested that the values of ICl and surface area were largest during interphase and smallest during M-phase of each cell cycle. This hypothesis was supported by an experiment in which entry into M-phase was blocked in fertilized eggs by inhibiting protein synthesis. This prevented the decreases of ICl and surface area but allowed the increases to occur normally. Patterns of change in ion currents are current specific and, as is the case with ICl, are tightly correlated with developmental events.     

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

Essential role of growth factor receptor-mediated signal transduction through the mitogen-activated protein kinase pathway in early embryogenesis of the echinoderm

In this study it was shown that growth factor receptors (GFR) play a crucial role in early embryogenesis of the echinoderms Hemicentrotus pulcherrimus and Clypeaster japonicus by transmitting signals to the mitogen-activated protein kinase (MAPK) pathway. The phosphorylation ratio of extracellular signal-regulated kinase 1 (ERK1) changed dynamically during early embryogenesis and showed a peak at the swimming blastula (sBl) stage. Suramin, an inhibitor of GFR, when applied during the sBl stage perturbed morphogenesis, including primary mesenchyme cell (PMC) migration, cell proliferation, archenteron elongation, spiculogenesis, pigment cell differentiation and phosphorylation of myosin light chains (MLC). Genistein, a receptor-type protein tyrosine kinase inhibitor, severely inhibited PMC migration, gastrulation and the phosphorylation of MLC. Manumycin A, a Ras inhibitor, inhibited spiculogenesis and invagination. PD98059, a MAPK/ERK kinase inhibitor, perturbed early PMC migration and pigment cell differentiation, but not spiculogenesis and gastrulation (although these two events were significantly delayed). PMC ingression was not perturbed by genistein, suramin, manumycin A or PD98059. All of the inhibitors perturbed the phosphorylation of ERK1, which was completely restored by exogenous platelet-derived growth factor (PDGF)-AB. PDGF-AB also partially restored elongation of the archenteron by restoring cell proliferation that had been perturbed by suramin.     

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo.

Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell-echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate.