BMP regulates vegetal pole induction centres in early xenopus development

BACKGROUND: Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. RESULTS: The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. CONCLUSIONS: These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.     

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.     

The origins of primitive blood in Xenopus: implications for axial patterning

The marginal zone in Xenopus laevis is proposed to be patterned with dorsal mesoderm situated near the upper blastoporal lip and ventral mesoderm near the lower blastoporal lip. We determined the origins of the ventralmost mesoderm, primitive blood, and show it arises from all vegetal blastomeres at the 32-cell stage, including blastomere C1, a progenitor of Spemann's organizer. This demonstrates that cells located at the upper blastoporal lip become ventral mesoderm, not solely dorsal mesoderm as previously believed. Reassessment of extant fate maps shows dorsal mesoderm and dorsal endoderm descend from the animal region of the marginal zone, whereas ventral mesoderm descends from the vegetal region of the marginal zone, and ventral endoderm descends from cells located vegetal of the bottle cells. Thus, the orientation of the dorsal-ventral axis of the mesoderm and endoderm is rotated 90( degrees) from its current portrayal in fate maps. This reassessment leads us to propose revisions in the nomenclature of the marginal zone and the orientation of the axes in pre-gastrula Xenopus embryos.     

The radon therapy: radon inhalation spa in Kowary

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90 degrees with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35-38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90 degrees rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90 degrees, but the fate of the blastomeres did not simply show a 90 degrees switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position: (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

Commitment to X inactivation precedes the twinning event in monochorionic MZ twins

Midkine (MK) is a heparin-binding growth factor that has been implicated in neural survival and differentiation, fibrinolysis, and carcinogenesis. It is expressed in the nervous system during early Xenopus development. In the present study, we demonstrated that injection of vegetal blastomeres with Xenopus MK at the 8-cell stage results in incomplete invagination. In the case of dorsal vegetal injection, hypertrophic neural tissue is produced. Animal caps isolated from embryos that have been injected with Xenopus MK and cultured with activin do not elongate, and all mesoderm markers examined, including both head and trunk/tail ones, are greatly diminished. In contrast, head-specific neural markers, XANF-1 and Xotx2, are induced, while trunk/tail neural markers, XlHbox6 and F-spondin, are decreased. Moreover, MK showes the same effects in animal caps injected with Xenopus Smad2 mRNA.     

GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.     

Localization of mitochondrial large ribosomal RNA in germ plasm of Xenopus embryos

In Xenopus, factors with the ability to establish the germ line are localized in the vegetal pole cytoplasm, or germ plasm, of the early embryo [1-3]. The germ plasm of Xenopus, and of many other animal species including Drosophila, contains electron-dense germinal granules which may be essential for germ-line formation [4-5]. Several components of the germinal granules have so far been identified in Drosophila [6-10]. One of these is mitochondrial large ribosomal RNA (mtlrRNA), which is present in the germinal granules (polar granules) during the cleavage stage until the formation of the germ-line progenitors or pole cells [8-9]. MtlrRNA has been identified as a factor that induces pole cells in embryos that have been sterilized by ultraviolet radiation [11]. The reduction of mtlrRNA in germ plasm by injecting anti-mtlrRNA ribozymes into embryos leads to the inability of these embryos to form pole cells [12]. These observations clearly show that mtlrRNA is essential for pole cell formation in Drosophila. Here, we report that mtlrRNA is enriched in germ plasm of Xenopus embryos from the four-cell stage to the blastula. Furthermore, our electron microscopic studies show that this mtlrRNA is present in the germinal granules during these stages. Thus, mtlrRNA is a common component of germinal granules in Drosophila and Xenopus, suggesting that the mtlrRNA has a role in germ-line development across phylogenetic boundaries.     

A role for Siamois in Spemann organizer formation

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.     

Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1

Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on development, and isolated animal caps are not mesodermalized. It is shown that Vg1 protein fails to form dimers and is not processed to release the putative bioactive domain. Furthermore it is shown that the N-terminal signal peptide of Vg1 is not cleaved following translocation into the ER, which may explain the failure of this protein to dimerize. To explore the role of Vg1 in amphibian development, a fusion protein has been made of the preproregion of Xenopus bone morphogenetic protein-4 and the putative bioactive C-terminal domain of Vg1. This fusion protein forms dimers and the C-terminal domain of Vg1 is secreted. Injection of this construct into Xenopus embryos induces the formation of a second dorsal axis and isolated animal caps are mesodermalized. The results are consistent with a role for Vg1 in mesoderm induction during Xenopus development.     

The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.     

Comparison of monolayer and bilayer plates used in antibiotic assay

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.     

Cell-cell association directed mitotic spindle orientation in the early development of the marine shrimp Sicyonia ingentis

During early cleavages of Sicyonia ingentis embryos, mitotic spindle orientations differ between blastomeres and change in a predictable manner with each successive mitosis. From 2nd through 7th cleavages, spindles orient at a 90 degrees angle with respect to the spindle of the parent blastomere. Thus, spindle orientation is parallel to the cleavage plane that formed the blastomere. To determine if specific spindle orientations were intrinsic properties of individual blastomeres, we altered blastomere associations and asked how mitotic spindle orientation was affected in successive cleavages using laser scanning confocal microscopy. Linear embryos were constructed by dissociating 4-cell embryos and recombining the blastomeres in a linear array. The ensuing cleavage (3rd embryonic cleavage) of these linear embryos was parallel to the long axis of the embryo, resulting in four parallel pairs of blastomeres which lay in a common plane that was parallel to the substratum. The 4th cleavage produced a linear embryo with the 16 blastomeres arranged in four parallel quartets. Then, in preparation for 5th cleavage, spindles oriented at a 45 degrees angle (not parallel as in normal development) with respect to the previous cleavage plane. When 8-cell linear embryos were separated into linear half-embryos, subsequent spindle orientations were not like those observed for intact 8-cell linear embryos, but rather regressed to the orientation seen in 4-cell linear embryos. We suggest that the reorientation of mitotic spindles during early cleavage of S. ingentis is neither an intrinsic property nor age dependent, but rather is cell contact related. Further, these results in conjunction with observations of non-manipulated embryos suggest that spindle poles (centrosomes) avoid cytoplasmic regions adjacent to where there is cell-cell contact during early development.     

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.     

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.     

Progressive effect of alpha-phenyl-N-tert-butyl nitrone (PBN) on rat embryo development in vitro

In the present study we demonstrated the effects of the spin-trapping agent alpha-phenyl-N-tert-butylnitrone (PBN) on the in vitro development of rat embryos at the early stage. In rat embryos, PBN increased the speed of the first cleavage and had no toxicity during pregnancy after embryo culture. These results showed that reactive oxygen species (ROIs) that were formed by activating molecular oxygens through redox reactions regulated the speed of development for early-stage embryos. Thus, PBN caused a decrease in the level of ROIs and toxicity and an in increase in the level of the development of rat embryos. On the other hand, PBN could not decrease the 2-cell block in vitro nor increase the blastulation rate, in contrast to the fact that a scavenger of superoxide anions, SOD, is effective in doing so for mouse embryos. From these results it was concluded that free radicals play an important role in the in vitro development of rat embryos at the early stage, but play no role in the decrease of the 2-cell block or their blastulation rate. It should be noted that PBN had no toxicity for embryonic development at the 2-cell stage.     

Induction of a secondary body axis in Xenopus by antibodies to beta-catenin

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The development of excitable cells is characterized by highly organized patterns of expression of ion channels. During the terminal differentiation of Xenopus muscle somites, potassium currents are expressed first just after Stage 15 (early-mid neurula), following a long period during which no voltage-dependent currents can be detected in any cell in the dorsal embryo. We have investigated whether early expression of a foreign delayed rectifier potassium channel may affect this endogenous pattern of electrical development. We injected the purified cRNA of the mammalian brain Shaker-like potassium channel, Kv1.1, into fertilized Xenopus eggs. The resulting currents were analyzed in blastomeres during a 12-hr period prior to Stage 15 and in differentiating muscle cells after Stage 15. In injected embryos, a high fraction of blastomeres expressed a delayed rectifier-type current. The Kv1.1 current could be distinguished from the endogenous muscle delayed potassium current (IK,X) by its very different voltage dependence. Separation of currents based on this difference indicated that, in injected embryos, IK,X appeared much earlier in development than in control embryos. Furthermore, even in cells which expressed solely Kv1.1-type current, the sensitivity of the current to dendrotoxin declined dramatically during development, approaching that of IK,X. These data suggest an interaction between Kv1.1 and endogenous channel subunits, and/or modification of the Kv1.1 protein by the embryonic cells in ways not seen in Xenopus oocytes or mammalian cell lines.     

An EORTC international multicenter randomized trial (EORTC number 19923) comparing two dosages of liposomal amphotericin B for treatment of invasive aspergillosis

Experiments were performed to determine the actions of recombinant bovine interleukin-1beta (IL-1beta) on the growth of preimplantation embryos. In the first series of studies, IL-1beta was added at 8-10 h after insemination, and the percentage of oocytes developing to the blastocyst stage was evaluated. IL-1beta increased development to the blastocyst stage when embryos were cultured at high density ( approximately 25-30 embryos/drop) but decreased or had no effect on development when cultured at low density ( approximately 10 embryos/drop). Thus, the positive effect of IL-1beta depends upon some other embryo-derived product. The effect of IL-1beta on embryonic development was maintained in completely denuded embryos, indicating that cumulus cells do not mediate the actions of IL-1beta. Maximum development of embryos cultured at approximately 25-30/drop occurred at 0.1-1 ng/ml; 10 ng/ml was less effective. Addition of IL-1beta to groups of approximately 25-30 embryos/drop at 8-10 h after insemination also increased embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached the 9- to 16-cell stage. However, IL-1beta had no effect on the proportion of blastocysts when added at Day 5 postinsemination. Thus, IL-1beta probably acts to increase blastocyst numbers by exerting actions on embryo growth before Day 5. In contrast to its effect on embryos, addition of IL-1beta during oocyte maturation did not affect cumulus expansion, cleavage rate of oocytes, or subsequent development to the blastocyst stage. In conclusion, IL-1beta can modulate growth of bovine embryos at early stages of development in a manner dependent upon embryo density.