Delayed transition to new cell fates during cellular reprogramming

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.     

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

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

Regionalized Cell Division during Sea Urchin Gastrulation Contributes to Archenteron Formation and Is Correlated with the Establishment of Larval Symmetry

During gastrulation of the sea urchin, Lytechinus variegutus there is localized proliferation of cells in the vegetal plate region prior to its invagination. Cell counts show that during gastrulation the number of cells per embryo increases 60% from 1025 to 1640. Measurements of cell volumes suggest that some growth may follow these divisions. Feulgen staining shows that the greatest mitotic activity throughout gastrulation occurs in the vegetal plate region. Labelling embryos with 3H-thymidine reveals that incorporation in the vegetal plate is confined to cells that encircle the base of the archenteron. Pulse-chase experiments indicate that these labelled cells contribute descendants to the vegetal half of the archenteron. Additionally, 3-dimensional reconstructions of vegetal regions at different stages reveal that by the end of gastrulation two bilateral clusters of labelled cells lie at the future sites of the post-oral arms of the pluteus larva, thus marking the axes of bilateral and dorso-ventral symmetry. Our findings suggest that two of the principal events of sea urchin gastrulation — the formation of the archenteron and the establishment of symmetry in the larva — are accompanied by distinct patterns of cell division.     

Characterization and expression of two matrix metalloproteinase genes during sea urchin development

Matrix metalloproteinases (MMPs) play an essential role in a variety of processes in development that require extracellular matrix remodeling and degradation. In this study, we characterize two MMPs from the sea urchin Strongylocentrotus purpuratus. These clones can both be identified as MMPs based on the presence of conserved domains such as the cysteine switch, zinc-binding, and hemopexin domains. In addition, both of these genes contain consensus furin cleavage sites and putative transmembrane domains, classifying them as membrane-type MMPs. We have named these clones SpMMP14 and SpMMP16 based on the vertebrate MMPs with which they share the greatest similarity. SpMMP14 is expressed in all cells from the egg to mesenchyme blastula stage embryo. Expression of this gene is strongest in the animal and vegetal poles early in gastrulation and in the animal pole only later in gastrulation. SpMMP16 is expressed at low levels in eggs. Expression of SpMMP16 becomes more pronounced in the vegetal pole region at the blastula and mesenchyme blastula stages and becomes confined to vegetal pole descendants, such as pigment cells, later in development. In the future, we hope to learn more about the possible functions of these genes in sea urchin development.     

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.     

EFFECT OF THE IMPase INHIBITOR L690,330 ON SEA URCHIN DEVELOPMENT

A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5mm on. Concentrations lower than 3.5mm permitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal—vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.     

STUDIES ON UNEQUAL CLEAVAGE IN SEA URCHINS I. MIGRATION OF THE NUCLEI TO THE VEGETAL POLE

It has been known for nearly a century that at the 16-cell stage of sea urchin embryos, the animal 4 cells divide equally and horizontally, whereas the vegetal 4 cells cleave unequally and practically vertically into macromeres and micromeres. Recently, more careful observations were made on the process of micromere formation and it has been revealed that a primary cause for the inequality lies in the migration of the 4 vegetal nuclei to the vegetal pole of the embryo which brings about excentricity of the mitotic apparatus. Records of this phenomenon are given in the present paper.     

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.     

Cell movements in the sea urchin embryo.

Recent studies show that gastrulation in the sea urchin embryo involves movement of cells over the blastopore lip (involution). Some cells in the vegetal plate of the late blastula become bottle-shaped but they play a limited role in gastrulation. The functions of specific integrins, regulators of cell-cell adhesion, and extracellular matrix components in gastrulation are currently being analyzed. In addition, light-microscopic studies continue to provide a unique picture of dynamic cell behavior in vivo.     

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.     

LvNotch signaling plays a dual role in regulating the position of the ectoderm-endoderm boundary in the sea urchin embryo

The molecular mechanisms guiding the positioning of the ectoderm-endoderm boundary along the animal-vegetal axis of the sea urchin embryo remain largely unknown. We report here a role for the sea urchin homolog of the Notch receptor, LvNotch, in mediating the position of this boundary. Overexpression of an activated form of LvNotch throughout the embryo shifts the ectoderm-endoderm boundary more animally along the animal-vegetal axis, whereas expression of a dominant negative form shifts the border vegetally. Mosaic experiments that target activated and dominant negative forms of LvNotch into individual blastomeres of the early embryo, combined with lineage analyses, further reveal that LvNotch signaling mediates the position of this boundary by distinct mechanisms within the animal versus vegetal portions of the embryo. In the animal region of the embryo, LvNotch signaling acts cell autonomously to promote endoderm formation more animally, while in the vegetal portion, LvNotch signaling also promotes the ectoderm-endoderm boundary more animally, but through a cell non-autonomous mechanism. We further demonstrate that vegetal LvNotch signaling controls the localization of nuclear beta-catenin at the ectoderm-endoderm boundary. Based on these results, we propose that LvNotch signaling promotes the position of the ectoderm-endoderm boundary more animally via two mechanisms: (1) a cell-autonomous function within the animal region of the embryo, and (2) a cell non-autonomous role in the vegetal region that regulates a signal(s) mediating ectoderm-endoderm position, possibly through the control of nuclear beta-catenin at the boundary.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

Heparin as a possible initiator of genomic RNA synthesis in early development of sea urchin embryos

In the early development of sea urchins, the uptake of sulfate ions into the embryo becomes prominent concurrent with the beginning of gastrulation, and the incorporated sulfate is invariably found in the heparin fraction.     

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.     

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.     

云南油杉原胚发育过程中组织化学的变化

应用组织化学方法对云南油杉(Keteleeria evelyniana Mast)受精前后,原胚及幼胚形成早期细胞内的DNA、RNA、碱性蛋白质,酸性蛋白质及多糖物质进行了观察。结果表明,DNA在卵核受精前后为孚尔根弱正反应。在原胚及早期幼胚发育过程中,胚原细胞核DNA含量恢复正常,RNA及酸性蛋白质含量均较丰富,特别是在胚原细胞内。新细胞质中碱性蛋白质呈负反应,而DNA、RNA、酸性蛋白质及多糖物质均呈现正反应。