Detection of endothelin-1 and its receptors in rat liver endothelial cells by in situ reverse transcriptase-polymerase chain reaction

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.     

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.     

Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles

In all animals so far tested, removing either pole of the undivided egg prevents normal development: embryos may arrest early, lack organs, or the adults may be sterile. These experiments have shown that spatial patterning of the egg is of utmost importance for subsequent development. However, the significance of spatial patterning in mammalian eggs is still controversial. To test the importance of egg polarity in the mouse a substantial amount of material either from the animal (polar body-associated) or the vegetal (opposite) pole of the fertilised egg was removed. One pole of the egg was cut away manually with a glass needle and the eggs were allowed to develop in vitro. Both kinds of surgical operation permit the development of blastocysts, which, after transfer to the uteri of pseudo-pregnant foster mothers, can produce viable offspring. Furthermore, these develop into fertile adult mice. I conclude that mouse eggs have no essential components that are localised uniquely to the animal or the vegetal pole and, therefore, do not rely for their axial development on maternal determinants that are so localised in the fertilised egg. Thus the mammalian egg appears to be very unusual in the animal kingdom in that it establishes the embryonic axes after the zygote has begun development.     

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.     

Localized maternal proteins in Xenopus revealed by subtractive immunization

It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.     

Dichotic pitches as illusions of binaural unmasking. I. Huggins' pitch and the "binaural edge pitch"

The two most salient dichotic pitches, the Huggins pitch (HP) and the binaural edge pitch (BEP), are produced by applying interaural phase transitions of 360 and 180 degrees, respectively, to a broadband noise. This paper examines accounts of these pitches, concentrating on a "central activity pattern" (CAP) model and a "modified equalization-cancellation" (mE-C) model. The CAP model proposes that a dichotic pitch is heard at frequency f when an individual across-frequency scan in an interaural cross-correlation matrix contains a sharp peak at f. The mE-C model proposes that a dichotic pitch is heard when a plot of interaural decorrelation against frequency contains a peak at f. The predictions of the models diverge for the BEP at very narrow transition bandwidths: the mE-C model predicts that salience is sustained, while the CAP model predicts that salience declines and that the dominant percept is of the in-phase segment of the noise. Experiment 1 showed that the salience of the BEP was sustained at the narrowest bandwidths that could be generated (0.5% of the transition frequency). Experiment 2 confirmed that the pitch of a BEP produced by a 0.5% transition bandwidth was close to the frequency of the transition band. Experiment 3 showed that pairs of simultaneous narrow 180-degree transitions, whose frequencies corresponded to vowel formants, were perceived as the intended vowels. Moreover, the same vowels were perceived whether the in-phase portion of the noise lay between the two transition frequencies or on either side of them. In contrast, different patterns of identification responses were made to diotic band-pass and band-stop noises whose cutoff frequencies corresponded to the same formants. Thus, the vowel-identification responses made to the dichotic stimuli were not based on hearing the in-phase portions of the noise as formants. These results are not predicted by the CAP model but are consistent with the mE-C model. It is argued that the mE-C model provides a more coherent and parsimonious account of many aspects of the HP and the BEP than do alternative models.     

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.     

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.     

Provisional bilateral symmetry in Xenopus eggs is established during maturation

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.     

The fertilization potential provides a fast block to polyspermy in lamprey eggs

At fertilization, the membrane potential of the egg of the lamprey, Lampetra japonica, shifted rapidly from its resting value of -12 to +36 mV and gradually returned to about the same resting level (fertilization potential). The amplitude of depolarization was influenced by the external Cl- concentration and by an anion channel blocker, DIDS, indicating that the positive shift of membrane potential resulted from Cl- efflux. A similar change in membrane potential (activation potential) was observed when the unfertilized egg was pricked with a fine needle or treated with A23187 to induce parthenogenetic activation. Pricking at the animal pole region (predetermined site for sperm entry) resulted in the occurrence of an immediate activation potential and the initiation of cortical granule exocytosis. A time lag between the pricking and the occurrence of the activation potential was observed when the egg was pricked at a distance from the animal pole. In this instance, the activation potential was produced immediately before the propagating cortical granule exocytosis initiated at the pricked site reached the animal pole region. Sperm-egg fusion was blocked in eggs voltage-clamped at +20 to +40 mV and inseminated, whereas it took place in eggs clamped at -60 to 0 mV. However, most eggs clamped at +20 to +40 mV did activate, indicating that the voltage dependence of egg activation differs from that of sperm-egg fusion. Although eggs voltage-clamped at negative membrane potentials permitted multiple sperm to fuse with the egg plasma membrane, the nucleus of the fused sperm did not necessarily enter the ooplasm. We conclude that: (1) A fast electrical block against polyspermy operates in this species and is effective for about 160 sec of the onset of the positive shift; (2) the opening of Cl- channels is responsible for the potential change; (3) the channels are largely localized in the animal pole region; (4) during voltage clamp at positive potentials, eggs can be activated without sperm-egg fusion; and (5) during voltage clamp at negative potentials, sperm-egg fusion occurs, but sperm entry into the egg cytoplasm does not always proceed.     

Relative changes in F-actin during the first cell cycle: evidence for two distinct pools of F-actin in the sea urchin egg

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.     

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.     

Na is essential for activation of the inseminated sea urchin egg

The presence of at least 2.5 mM Na during the first several min after insemination is required for the activation of sea urchin eggs. Of those chemical species examined that exist entirely as cations, and which did not activate the unfertilized egg, only Li substitutes for Na. Ammonium and other amines, with pKa values between 9 and 10.8, in the complete absence of Na (a) can induce nuclear activation of unfertilized eggs, and (b) permit the development of the early fertilized egg through the stage that normally requires Na.     

FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction

Normal pattern formation during embryonic development requires the regulation of cellular competence to respond to inductive signals. In the Xenopus blastula, vegetal cells release mesoderm-inducing factors but themselves become endoderm, suggesting that vegetal cells may be prevented from expressing mesodermal genes in response to the signals that they secrete. We show here that addition of low levels of basic fibroblast growth factor (bFGF) induces the ectopic expression of the mesodermal markers Xbra, MyoD and muscle actin in vegetal explants, even though vegetal cells express low levels of the FGF receptor. Activin, a potent mesoderm-inducing agent in explanted ectoderm (animal explants), does not induce ectopic expression of these markers in vegetal explants. However, activin-type signaling is present in vegetal cells, since the vegetal expression of Mix.1 and goosecoid is inhibited by the truncated activin receptor. These results, together with the observation that FGF is required for mesoderm induction by activin, support our proposal that a maternal FGF acts at the equator as a competence factor, permitting equatorial cells to express mesoderm in response to an activin-type signal. The overlap of FGF and activin-type signaling is proposed to restrict mesoderm to the equatorial region.     

Analysis of intracellular trafficking and interactions of cytoplasmic HIV-1 Rev mutants in living cells

Single blastomeres of the sixth-cleavage veg1 and veg2 tiers of Strongylocentrotus purpuratus embryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1 clones consistently contributed more cells to endoderm than aboral veg1 clones. Oral veg1 clones extended along the archenteron up to the foregut region, while aboral veg1 clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1 allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate marker Endo16 were more precisely determined by combining lineage tracer injection with whole-mount in situ hybridization. Endo16 expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to the Endo16 domain occurs in advance of the actual invagination of those cells. During the blastula stages Endo16 expression expands radially until all cells in the veg2 lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only the Endo16-expressing cells of the veg2 lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion of Endo16 into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.     

The 350-kDa sea urchin egg receptor for sperm is localized in the vitelline layer

Previous studies have established by several methods that the 350-kDa egg receptor for sperm is localized on the plasma membrane-vitelline layer complex of the egg of the sea urchin Strongylocentrotus purpuratus. In addition, it has been found that molecules which are cross-reactive with anti-receptor antibody are present in the cortical granules located at the inner face of the plasma membrane. The objective of this study was to define more precisely the locale of the cell surface receptor. We have found that following fertilization, the immunoreactive receptor initially found on the egg surface moved to the fertilization envelope (FE) and then disappeared in parallel with the loss of sperm binding activity. A cross-linked, high-molecular-weight derivative of soybean trypsin inhibitor (hMW-SBTI) which was unable to pass through the elevating FE blocked the loss of both immunoreactivity and the sperm binding activity of the FE, but did not inhibit the vitelline delaminase activity that has been implicated in FE formation. Western blot analysis following SDS-PAGE of the proteins of the FE isolated in the presence of hMW-SBTI and benzamidine revealed the presence of the 350/320-kDa proteins which cross-reacted with anti-receptor antibody. Experiments in which molecules on the surface of unfertilized eggs were labeled with biotin and traced after FE formation revealed that a significant portion of the 350/320-kDa glycoproteins that were incorporated into the FE originated from the cell surface, rather than from the cortical granules. These findings provide strong evidence that in unfertilized eggs the egg receptor for sperm exists as part of the protein complex known as the vitelline layer which serves as a precursor of the FE. Evidence is presented indicating that some of the receptor in the vitelline layer is cryptic and a possible function for this cryptic form of the receptor is proposed.     

Spatial distribution of collagen type I mRNA in Paracentrotus lividus eggs and embryos

We have identified the presence of type I collagen (COLL1alpha) mRNA in Paracentrotus lividus unfertilized egg, indicating a maternal origin of this mRNA. By in situ whole mount hybridization the spatial distribution of COLL1alpha mRNA in egg and embryo at different developmental stages was established. Moreover, the presence of COLL1alpha gene in Paracentrotus lividus genome was analyzed by Southern blot experiments. The localization pattern indicates that the maternal mRNA is placed in the fertilized egg in a fixed position, relative to the embryonic axes. Furthermore, the embryonic expression is spatially restricted during development, suggesting involvement in sea urchin embryo cell specification events. The presence of two bands in Southern blot hybridization may indicate that two genes specific for COLL1alpha are present in the sea urchin genome.     

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.     

Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum

In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.