Pregastrula Stage Research Articles

Angiomotin Regulates Visceral Endoderm Movements during Mouse Embryogenesis

In pregastrula stage mouse embryos, visceral endoderm (VE) migrates from a distal to anterior position to initiate anterior identity in the adjacent epiblast [1, 2]. This anterior visceral endoderm (AVE) is then displaced away from the epiblast by the definitive endoderm to become associated with the extra-embryonic ectoderm and subsequently contributes to the yolk sac. Little is known about the molecules that regulate this proximal displacement. Here we describe a role for mouse angiomotin (amot) in VE movements. amot expression is initially detected in the AVE and subsequently in the VE associated with the extra-embryonic ectoderm. Most amot mutant mice die soon after gastrulation with distinct furrows of VE located at the junction of the embryonic and extra-embryonic regions. Mutant analysis suggests that VE accumulation in these furrows is caused by defects in cell migration into proximal extra-embryonic regions, although distal-to-anterior movements associated with the epiblast, definitive endoderm formation, and anterior specification of the epiblast appear to be normal. These results suggest that amot acts within subregions of the VE to regulate morphogenetic movements that are required for embryo viability.

Expression and regulation of a netrin homologue in the zebrafish embryo

Proteins of the Netrin family have been implicated in axon guidance in both C. elegans and vertebrates. Here, we report the cloning and expression analysis of a zebrafish netrin homologue ( net1). net1 is expressed in the floor plate and the anterior ventral neural tube. Its expression is ectopically induced by misexpression of sonic hedgehog ( shh) and a dominant negative mutant of the regulatory subunit of protein kinase A (dnReg). Ectopic activation of net1, however, is restricted to distinct regions in the brain. Upon overexpression of shh or dnReg in cyclops mutants, which have strongly impaired net1 expression in the ventral neural tube, rescue of net1 expression was observed in the brain but not in the spinal cord. Ectopic expression of dnReg and Shh protein can be detected at high levels throughout injected embryos from pre-gastrula stages onwards suggesting that the competence of the neural plate to respond to Shh signalling activity differs regionally. Similar to net1, axial, the zebrafish homologue of mammalian HNF3β, which is also expressed along the ventral neural tube, is ectopically induced in the brain of embryos injected with dnReg mRNA. Neurons differentiate normally within domains of ectopic net1 and axial expression. Thus, dorsal neuronal differentiation appears to be unaffected despite co-expression of a gene program specific for the ventral neural tube. This also suggests that these ectopically expressing regions have not differentiated into floor plate.

Expression of exogenous genes in Xenopus oocytes, eggs, and embryos.

Molecular biology of gene expression in early amphibian embryogenesis began in 1964, when undegraded, as opposed to alkaline-hydrolyzed, PCA (perchloric acid), or TCA (trichloroacetic acid)-degraded, RNAs were extracted by Brown and Littna (, , ), Shiokawa and Yamana (,), and Woodland and Gurdon () from amphibian embryos using phenol methods. From these early studies, especially from those of Don Brown, it was realized that amphibian embryos exhibit quite unusual RNA synthetic patterns; the pattern of active 4S RNA (mainly tRNA) synthesis with no rRNA synthesis in pregastrula stages and the pattern of gradually increasing rRNA synthesis in post-gastrular stages. It was then reported in 1982 by Newport and Kirschner (,) that in Xenopus embryogenesis, large changes called midblastula transition (MBT) take place at the 12th cleavage, which include the appearance of Gl phase in cell cycle, onset of gene expression from zygotic nuclei, and acquisition of cellular motility (,). We then found in 1987 that Xenopus embryogenesis consists of three different phases with respect to the RNA synthetic pattern. The phase of a low level (on a per-embryo but not necessarily on a per-cell basis) of mRNA synthesis (pre-MBT stage), the phase of extremely active tRNA synthesis (both on per-cell and per-embryo bases) (MBT stage), and the phase of a nearly constant level (per cell) of rRNA synthesis (post-MBT stage) (, , , ).

DNA methylation in early development.

Several lines of evidence strongly suggest that DNA methylation is involved in embryo development. Perhaps the most direct evidence comes from experiments with methyltransferase deficient mice. Embryos that express low levels of the maintenance methyltransferase do not develop to term and die at the 5 to 20 somite stage corresponding to the level of the enzyme. In the mouse, dramatic methylation changes have been observed during the early steps of embryo development. Most genes are subject to a process of active demethylation starting promptly after fertilization. Except for a small number of methylated CpG sites in imprinted genes the vast majority of the sites are unmethylated by the stage of cavitation (16 cell). Such genome-wide demethylation may signify an erasure of epigenetic information originating in the highly differentiated gametes. This erasure may be essential for the establishment of a pluripotent state, before specific cell lineages can be determined. The process of laying down a new developmental program involves, initially, global de novo methylation at the stage of pregastrulation followed by gene specific demethylations associated with the onset of activity of these genes. CpG islands characteristic of housekeeping genes, appear to be protected from the global de novo methylation. An exception to this rule is the hypermethylation of CpG islands in X-linked housekeeping genes on the inactive X chromosome and of specific differentially methylated CpG sites in imprinted genes. Primordial germ cells escape the global de novo methylation which takes place at the pregastrula stage and undergo a very similar de novo methylation process in the differentiated gonads (15.5-18.5 days post coitum), forming the methylation patterns which are specific to the gametes. Specific demethylations then form a terminal methylation pattern which is then clonaly inherited in the soma and erased after fertilization.

A β1-integrin associated α-chain is differentially expressed during Xenopus embryogenesis

The antigen of mAb 2F10 was identified as a Xenopus β 1-integrin associated α-chain by the criteria (1) that it coprecipitates with anti β 1-antibody, (2) that it changes molecular mass upon reduction in a way that is characteristic for integrin α-chains and (3) that it is present on cell membranes. This α-chain, termed α 2F10, is found in small amounts in the pregastrula stages of Xenopus development and accumulates thereafter in the embryo. α 2F10 can be detected by immunofluorescence first at stage 17 of embryogenesis on the cell membranes of the sensorial layer of the ectoderm, the notochord and the endoderm. This characteristic pattern of distribution is maintained throughout the following embryonic st ages. Timed explantation experiments indicate that all cells of the pregastrula have the potency to express α 2F10. This potency becomes successively restricted during gastrulation to yield the ultimate pattern of expression.

Evidence that the border of the neural plate may be positioned by the interaction between signals that induce ventral and dorsal mesoderm.

In the early Xenopus embryo, a quadrant of endodermal cells that have descended from the vegetal dorsal localization in the zygote produces signals that pass into the animal hemisphere and induce dorsal mesoderm from the marginal zone. From the remaining three quadrants of the bordering endoderm, signals pass into the animal hemisphere and induce ventral mesoderm in the marginal region. There is evidence that suggests that these same mesoderm-inducing signals continue through the plane of the tissue of the animal hemisphere where they may at least begin the processes of neural and epidermal induction by changing the competence of the prospective ectodermal cells, and possibly influencing the early regional biasing of later expression of at least some gene products, such as Epi-1 whose expression in the future epidermal domain seems specified before gastrulation. We hypothesized that the interaction of the ventral and dorsal signals within the plane of the tissue of the animal hemisphere may position the border of the neural plate. If this is so, then transplantation into the animal pole of cells that signal induction of ventral mesoderm should drive the neural plate boundary back toward the blastopore and shorten the anterior-posterior axis. Removal of cells that induce ventral mesoderm should result in an axis that is longer than normal. Results of our experiments support these predictions. Also, by late pregastrula stage 9, increasing the ventral signals has no effect. Thus the evidence suggests that the position of the anterior neural plate boundary is established before gastrulation begins by the interaction of the signals that induce the ventral and dorsal mesoderm.

Change in the Activity of Na1 , K+ -ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development: (sea urchin embryo/Na+ , K+ -ATPase/RNA synthesis/ectodermal cell/actinomycin D).

The activity of ouabain-sensitive Na+ , K+ -ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo-wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre-gastrula stage and ectodermal cells at the late gastrula stage, the Na+ , K+ -ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells. Cycloheximide did not only block the activity increase in this period but also caused evident decrease in the activity in embryos at all examined stages. The activity increase in this period was strongly blocked by the treatment with actinomycin D, starting before the mesenchyme blastula stage, and was not seriously inhibited by the treatment starting at the mesenchyme blastula stage. The treatment starting at the initiation of gastrulation only slightly blocked further increase in the activity. Probably, an accumulation of mRNA encoding Na+ , K+ -ATPase occurs mainly in ectodermal cells and is completed up to the early gastrula stage.

Characterization of protein kinase C in early Xenopus embryogenesis

Recently, we presented evidence that protein kinase C (PKC) is involved in mediating the endogenous signals that induced competent Xenopus ectoderm to differentiate to neural tissue. We report here that PKC is already strongly activated in neural-induced ectoderm from midgastrula embryos and that this activation runs parallel with an increase in the level of inositol phosphates. We further identify several proteins that are phosphorylated, both in natural neural-induced ectoderm and in TPA-treated ectoderm, suggesting that they are phosphorylated through the PKC route. We found no major changes in PKC activity among different pregastrula stages, including the unfertilized egg. However, PKC isolated from animal, ectodermal cells is highly sensitive to Ca2+ and can be activated by low concentrations, (6-25 microM) of arachidonic acid, while PKC isolated from vegetal, endodermal cells is more insensitive to Ca2+ and cannot be activated by arachidonic acid. These results suggest that different PKC isozymes are present in animal and vegetal cells.

Fibroblast growth factor during mesoderm induction in the early chick embryo

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.

Synthesis of laminin-related polypeptides in oocytes, eggs and early embryos of the amphibian Pleurodeles waltlii.

In the amphibian Pleurodeles waltlii, lamininrelated polypeptides (amphibian-LN) are present in the extracellular matrix underlying the blastocoel roof of gastrulating embryos. Immunoprecipitation with affinity-purified anti-laminin antibodies demonstrated that amphibian-LN is synthesized in oocytes (from stage III onward), eggs and throughout early development. At the late blastula stage, when experiments were carried out with animal and vegetal halves, there were no regional differences in the pattern of amphibian-LN synthesis. The results obtained with transcription inhibitors suggest that throughout pregastrula stages, amphibian-LN is a translation product of stored maternal mRNA. Finally, having compared amphibian-LN and fibronectin synthesis, it is concluded that both extracellular glycoproteins have a common pattern of synthesis.

Effects of Various Antibiotics on the Excitability of Newt Ectodermal Cells in vitro: (amphibian embryos/epidermal action potential/cell culture/antibiotics).

With the addition of actinomycin D (AMD), cycloheximide (CH) or tunicamycin (TM) to a culture medium at 23°c, the epidermal action potentials (EAPs) of the cultured epidermal explants or monolayered cell colonies taken from newt embryos were examined for confirmation of their relationship to transcriptional and translational events. EAPs of ectodermal cells from the pregastrula were first recorded after 84 hr of monolayered cell culture in FCS-Steinberg's medium. The addition of 10 μg/ml CH or 1 μg/ml TM for 36 hr prevented the appearance of these potentials in cells precultured in FCS-Steinberg's medium for 60 hr. Ectodermal cells precultured in FCS-Steinberg's medium for 68 hr generated the EAPs after they were cultured in the same concentration of CH or TM for 28 hr. Thus, it appears that certain proteins and glycoproteins may be responsible for evoking the EAPs and that these molecules are synthesized at about 68 hr in culture following the pregastrula stage. Ectodermal cells precultured in FCS-Steinberg's medium for 48 hr generated EAPs after they were cultured in 0.5 μg/ml AMD for 72 hr. Cells precultured for 36 hr failed to produce the EAPs even when they were cultured in the same concentration of AMD for 84 hr.

Changes in Activity of Mitochondrial Enzymes during the Development of Xenopus laevis: (Mitochondria/emzymes/embryonic development/Xenopus).

Changes in activity of mitochondrial enzymes were studied during the embryonic development of Xenopus laevis. The following enzymes were determined: malate dehydrogenase (MDH), isocitrate dehydrogenase (NAD+ -dependent) (IDH), aspartate aminotransferase (GOT), cytochrome oxidase (COX), succinate dehydrogenase (SDH), rotenone-insensitive NADH cytochrome c reductase (NADH-red) and monoamine oxidase (MAO). IDH is constant throughout the period studied. COX and SDH, two enzymes of the inner membrane, are constant in pregastrula stages, and subsequently decrease significantly. MDH and NADH-red are highly active in the pregastrula stages and decline thereafter, while MAO is undetectable during early development and increases significantly only in the larvae. GOT increases during the cleavage stages, being most active in the gastrula stages, and decreases subsequently. The results are discussed in the sense of mitochondrial differentiation during the early development of the amphibian embryo.

Changes in the activities of mitochondrial carriers during the development of Xenopus laevis

The transport of the substrates phosphate, pyruvate, malate, fumarate, α-ketoglutarate, citrate, isocitrate and glutamate has been determined in mitochondria isolated from various developmental stages of Xenopus laevis. The carriers may be sub-divided into three groups with respect to the pattern of changes in activity: 1. 1. Pyruvate, tricarboxylate and glutamate/OH carriers, distinguished by low activities during the pregastrula stages and a subsequent increase in activity. 2. 2. Phosphate and α-ketoglutarate carriers, with a constant activity throughout development. 3. 3. Malate and fumarate carriers, highly active initially, but decreasing already after gastrulation. The results are discussed in relation to mitochondrial differentiation and the change from embryonic to adult metabolism.

Circus movements in dissociated cells in normal and hybrid frog embryos.

Circus movements, involving circumferential rotation of a hyaline cytoplasmic blister and endoplasmic flow, occur in EDTA-dissociated gastrula stage Rana pipiens embryos. Such cell movements occur in very few cells taken from pre-gastrula stage embryos. During gastrulation, there is a progressive increase in the proportion of a population of cells that is engaged in circus movements. Circus movements do not occur in dividing cells. Individual cells in culture, as well as small clusters of cells in vitro, are jostled about in an apparently aimless fashion over short distances by circus movements, although the translocation of masses of cells over long distances is substantially greater than the translocation of isolated cells. In an early gastrula stage normal embryo, cells from around the site of blastopore invagination are most active in circus movements. Cells taken from different stages of arrested hybrid embryos show variable depression in the formation of rotating hyaline blebs. Aggregates of cells from arrested hybrid embryos are also relatively immobile in culture. The morphogenetic significance of circus movements in normal embryos and gastrula-arrest hybrid embryos is discussed.

Membrane differentiation in the pregastrula of the teleost, Brachydanio rerio Hamilton-Buchanan (Teleostei: Cyprinidae). A scanning electron microscope study.

The pregastrula stages of the teleost, Brachydanio rerio, have been studied in intact or fractured eggs by scanning electron microscopy. The internal surface of the enveloping blastomeres was smooth, while the external surface was covered with cristae; these cristae disappeared during epiboly but reappeared when the cells were in mitosis. At the end of the morula stage, the smooth yolk cytoplasmic layer formed a ring of periblastic cristae; up to the late blastula stage that ring was attached to "star-like" complexes. Light and transmission electron microscopy images of these complexes are given and we propose hypotheses regarding their function. At first closely apposed, the deep cells seemed to be maximally dispersed during the mid-blastula stage. At the blastula stage, the yolk syncitium formed both enveloping and deep cells. This suggests a primordial role of the yolk syncitium layer in pregastrula differentiation. A new hypothesis concerning the functions of the membrane protrusions is proposed: they might play a role in the postmiotic reassociation of the blastomeres. We suggest that, during early development, the three modes of cell association, i.e. monolayer, mass aggregation, syncitium, might be correlated with the composition of the three media (perivitelline liquid, blastocoelic liquid, yolk) present at that time.

POLARITIES, CELL DIFFERENTIATION AND PRIMARY INDUCTION IN THE AMPHIBIAN EMBRYO

POLARITIES, CELL DIFFERENTIATION AND PRIMARY INDUCTION IN THE AMPHIBIAN EMBRYO

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Polypyrimidines, 50 to 200 nucleotides in length, have been found in the nuclear DNA of Strongylocentrotus purpuratus embryos. The distribution of lengths of pyrimidine runs resulting from depurination of main-density band sea urchin DNA, separated by CsCl gradient centrifugation, is the same as that obtained from the unfractionated DNA. When embryo DNA is separated into unique and repetitive sequence fractions, the polypyrimidines having more than 50 nucleotides are associated, for the most part, with the repetitive sequence fractions of the DNA. Removal of regularly spaced, naturally occurring, single-stranded regions (by digestion with Aspergillus oryzae S1 nuclease) from morula stage native DNA causes a partial depletion of polypyrimidines 60 to 80 nucleotides in length. Treatment of gastrula stage native DNA, which lacks single-stranded regions, with S1 nuclease does not alter the size pattern of polypyrimidines, compared with untreated embryo DNA. Removal of single-stranded regions from repetitive sequence morula stage DNA by treatment with S1 nuclease also causes a reduction in polypyrimidines 60 to 80 nucleotides in length. It is concluded that eukaryote-specific polypyrimidines, 50 to 200 nucleotides in length, are associated primarily with repetitive sequence DNA and that a portion of the isostichs (see Introduction) 60 to 80 nucleotides in length occur at nonrandom sites which are single-stranded during pre-gastrula stage development. This latter conclusion, and a previous finding that these single-stranded regions occur as staggered pairs (on opposite strands) at the center of native unit length sections of duplex morula stage DNA, suggests that the 60 to 80 nucleotide-length polypyrimidines may be in register with sites which demarcate bidirectional replication regions along the genome.

Variation in rDNA redundancy level and nucleolar organizer length in normal and variant lines of the Mexican axolotl.

ABSTRACT The level of redundancy of ribosomal genes, and the relationship of this level to nucleolar formation at different stages of embryonic development, have been examined in the Mexican axolotl, Ambystoma mexicanum. Individuals from 4 inbred stocks were examined, as well as descendants from 2 nucleolar variants which, in the heterozygous condition, are distinguished by exceptionally small nucleoli. Ribosomal RNA-DNA hybridization assays show that one of the 4 wild type lines has only about one-third as much ribosomal DNA (rDNA) as the other three. One of the nucleolar variants has the same level of rDNA as the larger wild-type level; the other variant has the same amount as the smaller ribosomal genome line. Both original nucleolar variants arose as F1, progeny of crosses between a large rDNA genome line and the small genome line. Cytological examination of pregastrula stage embryos from wild type and nucleolar variant lines show that the lengths of the nucleolar organizer regions (NOR) and the sizes of nucleoli formed, are directly correlated with the amount of rDNA present at the nucleolar locus. During gastrulation of the nucleolar variants, however, a transition appears to take place and the amount of rDNA ceases to be the determining factor in nucleolar size. After late gastrula, heterozygous progeny resulting from crosses of either large rDNA genome or small rDNA genome wild type individuals with either nucleolar variant line, have a small and a large nucleolus. The factor or factors associated with this apparent lack of competitive ability of the variant NOR, when opposed to a normal NOR, are unknown. It might be suggested that since the chromosomal alterations which produced the nucleolar variants in both cases eliminated the gene determining the dark colour pattern, they could at the same time have eliminated other genetic material.

Ribosomal RNA synthesis in pre- and postgastrula embryos of xenopus laevis

The synthesis of ribosomal RNA in pre- and postgastrula embryos of Xenopus laevis was examined using 3H-CH 3-methionine. No incorporation of label into ribosomal RNA is seen in pregastrula stages be it whole cell or ribosome extracts. These data support the observation that during early embryogenesis of Xenopus the synthesis of ribosomal RNA is under regulation.

Mouvements cellulaires dans le développement de l'embryon des amphibiens et des poissons

The study of the cellular movements during the pregastrula and gastrula stages, in amphibia and teleosts, shows resemblances and differences in connection with the structure of the egg. The entire egg of amphibia does not show any activity during the pregastrula stages; the teleost one is animated with complex movements, which are sometimes distinctly polarized. This activity appears in the cortical layer, in contact with the yolk. If it does not envelop pure cytoplasm only (embryonic shield) the cortex is inactive like the amphibia's. Thus the motor activity does not depend upon the cortex alone, but probably upon the interaction cortex/yolk. The invagination during gastrulation is not an infolding towards the depth of the egg-wall but brings in individual displacements of the blastomeres: the deep blastomeres proceed, the ones on the others, towards the blastocoel, and into that moving mass, the superficial blastomeres (flask cells), endowed with a polarized motor activity, go deep more quickly. During normal development, the yolk mass of the teleosts might be used as a support for the moving cells, but this part is not absolutely necessary as the invagination accomplishes itself in the expiants deprived from yolk. If the layer of superficial blastomeres (enveloping layer) is suppressed, the invagination cannot be carried out (teleosts). The epiboly is due to the autonomous intrinsic spreading force acquired by the cells of the animal hemisphere at the beginning of the gastrulation. In experimental conditions, a capacity of spreading, which can be compared to an epibolic movement, can be set in evidence in the centre of the pregastrulean egg. The epibolic capacity, in teleosts, is limited only to the cells of the enveloping layer, as it appears in the culture experiments. The realization of the epibolic movement, more complex in teleosts than in amphibia, results from the cooperation of force groups, respectively localized in the segmented and non-segmented portions (vitelline syncytium, perivitelline pellicle) of the egg. These two movements of invagination and epiboly are not connected and may be realized in gastrula separately. The mechanism of the morphogenetic movements sets the following problems: 1. 1. spontaneous mobilization of cells 2. 2. nature of the individual movements and coordination 3. 3. orientation of the movements. In conclusion, these questions are rapidly examined. It is suggested that the initiation of the activity and the orientation might be dependent upon secreted substances.