Changes in the Pattern of Intercellular Junctions during Early Embryogenesis of the Starfish, Asterias amurensis

Changes in the cellular adhesion pattern during the early embryogenesis of a starfish Asterias amurensis were examined using carboxyfluorescein (CF) dye as a probe. CF that was injected into one of the blastomeres at the 2- or 4-cell stage was in all cases restricted to the progeny cells of the CF-labelled blastomere. With the advancement of gastrulation, however, the injected dye was distributed not only to the progeny of the labelled blastomere, but also to cells that originated from non-injected blastomeres. At the beginning of mesenchyme cell release, the injected dye spread uniformly to most cells comprising the embryo. When one of the blastomeres situated in the vegetal hemisphere of an 8-cell embryo was labelled, the resulting embryo showed more intense fluorescence in the cells surrounding the archenteron than in the ectodermal layer, suggesting that the cells in ectodermal layer became associated more intimately or earlier than those surrounding the archenteron. Likewise, in double embryos formed by combining two denuded eggs, in which one egg had been labelled with CF, dye spread was observed when the ectodermal layer began to expand. The intercellular spread of CF dye in starfish embryo suggests that there is a dramatic change in the cellular adhesion pattern during the course of gastrulation.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Pattern regulation in defect embryos of Xenopus laevis

Defect embryos of 24 series were prepared by removing increasing numbers of blastomeres from an 8-cell embryo of Xenopus laevis. They were cultured and their development was examined macroscopically when controls reached a tailbud stage or later. Results show that most of defect embryos of 12 series develop normally, and some of them become normal frogs. Each of these defect embryos contain at least two animal blastomeres, one dorsal, and one ventral blastomere of the vegetal hemisphere. This suggests that a set of these four blastomeres of the three types is essential for complete pattern regulation.     

Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16-cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCI-treated whole embryos developed into exo-gastrulae but Hpoe restriction in ectoderm occurred in these exo-gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpress     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.     

The Determinant for Archenteron Formation in Starfish: Co-Culture of an Animal Egg Fragment-Derived Cell Cluster and a Selected Blastomere

A method of detecting blastomeres that carrying the determinant for archenteron formation was established, based on the reported localization of the determinant in the vegetal cytoplasm (17, 24). The essence of the method was to co-culture a selected blastomere with an animal egg fragment-derived cell cluster, so as to generate one joined embryo. The presence of the determinant in the blastomere was assessed by the formation of the archenteron in the developed joined embryos. The vegetal blastomeres that carried the determinant sometimes induced animal egg fragment-derived cells to form part of the archenteron.     

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.     

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.     

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Mesendoderm cells induce oriented cell division and invagination in the marine shrimp Sicyonia ingentis

The mesendoderm (ME) cells are the two most vegetal blastomeres in the early developing embryo of the marine shrimp Sicyonia ingentis. These two cells enter mitotic arrest for three cycles after the 5th cell cycle (32-cell stage) and ingress into the blastocoel at the 6th cycle (62-cell stage). Circumjacent to the ingressing ME cells are nine presumptive naupliar mesoderm (PNM) cells that exhibit a predictable pattern of spindle orientation into the blastopore, followed by invagination. We examined the role of ME cells and PNM cells in gastrulation using blastomere recombinations and confocal microscopy. Removal of ME progenitors prevented gastrulation. Removal of any other blastomeres, including PNM progenitors, did not interfere with normal invagination. Altered spindle orientations occurred in blastomeres that had direct contact with one of the ME cells; one spindle pole localized to the cytoplasmic region closest to ME cell contact. In recombined embryos, this resulted in an extension of the region of ME-embryo contact. Our results show that ME cells direct the spindle orientations of their adjacent cells and are consistent with a mechanism of oriented cell division being a responsible force for archenteron elongation.     

Lithium changes the ectodermal fate of individual frog blastomeres because it causes ectopic neural plate formation

Amphibian blastulae that are treated with lithium (Li) develop into embryos that consist almost exclusively of head structures. This dramatic change in embryogenesis may occur either because Li selectively kills trunk progenitors or because Li causes trunk progenitors to become head progenitors. To distinguish between these possibilities, we compared the fates of individual frog blastomeres between Li-treated embryos and normal embryos using lineage tracers. The results demonstrate that Li causes ventral midline cells, which normally populate large amounts of trunk, to produce many head structures, including the brain. Examination of fluorescently labeled clones in living Li-treated gastrulae shows that: (1) the ectodermal members of the clones migrate normally, and chordamesodermal involution begins normally; (2) the chordamesoderm's later involution is altered such that it is confined to the vegetal hemisphere; (3) accordingly, the neural plate forms in the vegetal hemisphere, circumscribing the blastopore, which normally gives rise to the cloaca; and (4) the ectodermal progeny of the ventral midline blastomeres that are near the blastopore populate the brain because they are induced by the stalled chordamesoderm to form part of the ectopic neural plate. These results demonstrate that Li, administered during a short developmental window at early cleavage stages, ultimately alters ectodermal fate because it changes the pattern of chordamesodermal involution during gastrulation, which in turn changes the site of neural plate formation.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

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.