Animal Pole Region Research Articles

Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish.

The vegetal pole cytoplasm represents a crucial source of maternal dorsal determinants for patterning the dorsoventral axis of the early embryo. Removal of the vegetal yolk in the zebrafish fertilised egg before the completion of the first cleavage results in embryonic ventralisation, but removal of this part at the two-cell stage leads to embryonic dorsalisation. How this is achieved remains unknown. Here, we report a novel mode of maternal regulation of BMP signalling during dorsoventral patterning in zebrafish. We identify Vrtn as a novel vegetally localised maternal factor with dorsalising activity and rapid transport towards the animal pole region after fertilisation. Co-injection of vrtn mRNA with vegetal RNAs from different cleavage stages suggests the presence of putative vegetally localised Vrtn antagonists with slower animal pole transport. Thus, vegetal ablation at the two-cell stage could remove most of the Vrtn antagonists, and allows Vrtn to produce the dorsalising effect. Mechanistically, Vrtn binds a bmp2b regulatory sequence and acts as a repressor to inhibit its zygotic transcription. Analysis of maternal-zygotic vrtn mutants further shows that Vrtn is required to constrain excessive bmp2b expression in the margin. Our work unveils a novel maternal mechanism regulating zygotic BMP gradient in dorsoventral patterning.

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis.

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.

AnkAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo

In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.

Ultrastructural study on the fertilisation process in sturgeon ( Acipenser), function of acrosome and prevention of polyspermy

Sturgeon gametes differ from other fish in that their spermatozoa possess acrosome with finger-like posterolateral projections, which undergo exocytosis and filament formation, whereas eggs possess numerous micropyles. The fertilisation process in Acipenser baerii was investigated by fluorescence and electron microscopy. A suitable activation solution containing 2.5 mM CaCl 2, 15 mM Tris, pH 10 was found for detailed description of acrosomal reaction. The acrosome reaction includes the formation of a spear-like fertilisation filament coming from three endonuclear canals and implantation fossa through the acrosomes. It can accelerate the process of polyspermy prevention. Another unique feature of the acrosome was an anchor-like opening of the posterolateral projections. Mature eggs of A. baerii possessed 2–10 micropyles in the animal pole region. The eggs consisted of three principal layers and an outermost jelly coat blocking micropyle, and a layer of cortical granules in unfertilised eggs. With the exposure to freshwater, the jelly like layer separated from the egg surface, whereas the cortical granules swelled. No change between the layers of fertilised and unfertilised eggs, apart from the generation of an increasing perivitelline space by dissolution of the cortical granules, had been observed after the fusion of spermatozoon with an egg. A fertilisation cone blocked a fusion of other spermatozoa with cytoplasmatic projection in the fertilised micropyle.

The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center

Two major signaling centers have been shown to control patterning of sea urchin embryos. Canonical Wnt signaling in vegetal blastomeres and Nodal signaling in presumptive oral ectoderm are necessary and sufficient to initiate patterning along the primary and secondary axes, respectively. Here we define and characterize a third patterning center, the animal pole domain (APD), which contains neurogenic ectoderm, and can oppose Wnt and Nodal signaling. The regulatory influence of the APD is normally restricted to the animal pole region, but can operate in most cells of the embryo because, in the absence of Wnt and Nodal, the APD expands throughout the embryo. We have identified many constituent APD regulatory genes expressed in the early blastula and have shown that expression of most of them requires Six3 function. Furthermore, Six3 is necessary for the differentiation of diverse cell types in the APD, including the neurogenic animal plate and immediately flanking ectoderm, indicating that it functions at or near the top of several APD gene regulatory networks. Remarkably, it is also sufficient to respecify the fates of cells in the rest of the embryo, generating an embryo consisting of a greatly expanded, but correctly patterned, APD. A fraction of the large group of Six3-dependent regulatory proteins are orthologous to those expressed in the vertebrate forebrain, suggesting that they controlled formation of the early neurogenic domain in the common deuterostome ancestor of echinoderms and vertebrates.

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.

Animal pole determinants define oral–aboral axis polarity and endodermal cell-fate in hydrozoan jellyfish Podocoryne carnea

Cnidarians, in contrast with bilaterians, are generally considered to exhibit radial symmetry around a single body axis (oral–aboral) throughout their life-cycles. We have investigated how the oral–aboral axis is established in the hydrozoan jellyfish Podocoryne carnea. Vital labeling experiments showed that the oral end of the blastula derives from the animal pole region of the egg as has been demonstrated for other cnidarian species. Gastrulation is restricted to the oral pole such that the oral 20% of blastula cells give rise to endoderm. Unexpectedly, bisection experiments at the 8-cell stage showed that animal regions are able to develop into normally polarized larvae, but that vegetal (aboral) blastomeres completely fail to develop endoderm or to elongate. These vegetal-derived larvae also failed to polarize, as indicated by a lack of oral-specific RFamide-positive nerve cells and a disorganized tyrosinated tubulin-positive nerve net. A different result was obtained following bisection of the late blastula stage: aboral halves still lacked the capacity to develop endoderm but retained features of axial polarity including elongation of the larva and directional swimming. These results demonstrate for the first time in a cnidarian the presence of localized determinants responsible for axis determination and endoderm formation at the animal pole of the egg. They also show that axial polarity and endoderm formation are controlled by separable pathways after the blastula stage.

Establishment of animal–vegetal polarity during maturation in ascidian oocytes

Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal–vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal–vegetal (a–v) axis to establish the embryonic (D–V, A–P) axes. To understand how this radial a–v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a–v axis occurs after maturation in several steps which take 3–4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians.

Cloning and developmental expression of a novel, secreted frizzled-related protein from the sea urchin, Strongylocentrotus purpuratus

Wnt proteins and their receptors, members of the frizzled protein family, play a key role in regulating a wide range of developmental processes. Recently, putative regulators of Wnt signaling known as secreted frizzled-related proteins (SFRPs) have been identified in several vertebrates. Here, we describe the cloning of a novel SFRP (suSFRP1) from the sea urchin, Strongylocentrotus purpuratus. SuSFRP1 contains a putative signal sequence, four cysteine-rich domains and a single Ig domain. The developmental expression of suSFRP1 mRNA is highly dynamic and can be separated into three phases: (1) abrupt accumulation in most or all cells of the embryo at the early blastula stage; (2) restriction of expression to the prospective endoderm and animal pole region of the gastrula; and (3) expression in prospective muscle cells of the coelomic pouches during late embryogenesis.

A novel POZ/zinc finger protein, champignon, interferes with gastrulation movements in Xenopus.

We have cloned a novel krüppel-like transcription factor of Xenopus that encodes POZ/zinc finger protein by expression cloning. Overexpression of mRNA resulted in interference with gastrulation. Because the injected embryo looks like a mushroom in appearance at the neurula stage, we have named this gene champignon (cpg). In cpg-injected embryos, the blastopore appeared normally, but regressed thereafter. The injected embryos then elongated along the primary dorsoventral axis during the tailbud stage. Histologic sections and reverse transcription-polymerase chain reaction analysis showed that cpg had no effect on the cell differentiation. The animal pole region of cpg-injected embryos was thick during the gastrula stage, and mesodermal cells remained in the marginal zone. Furthermore, neither Keller-sandwich explants nor activin-treated animal cap explants excised from cpg-injected embryos elongated. These results suggest that cpg acts as a potent inhibitor of cell migration and cell intercalation during gastrulation.

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca(2+) and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at </=40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6-dimethylaminopurine (6-DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short-lived protein kinase(s), sensitive to 6-DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Copyright 1999 Wiley-Liss, Inc.

Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs.

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal-vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.

Anterior structural defects by misexpression of Xgbx-2 in early Xenopus embryos are associated with altered expression of cell adhesion molecules.

The RNA of the noncluster homeobox gene, Xgbx-2, is localized during neurulation to a narrow band of tissue at the midbrain hindbrain boundary (anterior hindbrain). The localized expression of Xgbx-2 within the nervous system prompted us to assess its function during early development by injection of synthetic Xgbx-2 RNA into the animal pole region of both dorsal blastomeres at the four-cell stage. Injection of Xgbx-2 RNA leads to dose-dependent alterations in anterior dorsal structures. These defects include abnormal eye development including reduced and missing eyes, reduced or missing cement glands, and abnormal brain development. Additionally, coinjection with lineage label (either beta-galactosidase or green fluorescent protein) shows there is a dose-dependent misplacement of cells. These misplaced cells can be found in such locations as the blastocoele, gastrocoele, or ventricles in the brain. In some spawnings, misplaced cells are expelled from the embryo into the periviteline space. In general, the phenotype of Xgbx-2 RNA-injected embryos is strikingly similar to the phenotypes observed when dominant-negative RNA constructs of Ca2+-dependent cell-adhesion molecules are injected into similar regions of early embryos. Xgbx-2 misexpression enhanced the dissociation of animal hemisphere cells, and inhibited Ca2+-dependent cell adhesion in dissociated animal hemisphere cells in vitro. Additionally, when the expression of various calcium-dependent cadherins was tested, it was shown that misexpression of Xgbx-2 prevents N-cadherin expression during early neurulation. These observations suggest that the transcription factor, Xgbx-2, functions normally in the regionalization of the neural tube (specifically the anterior hindbrain) by regulating differential cell adhesion and subsequently cell identity.

Patterns of free calcium in zebrafish embryos.

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.

Anteroposterior patterning in the zebrafish, Danio rerio: an explant assay reveals inductive and suppressive cell interactions.

We report the first extended culture system for analysing zebrafish (Danio rerio) embryogenesis with which we demonstrate neural induction and anteroposterior patterning. Explants from the animal pole region of blastula embryos ('animal caps') survived for at least two days and increased in cell number. Mesodermal and neural-specific genes were not expressed in cultured animal caps, although low levels of the dorsoanterior marker otx2 were seen. In contrast, we observed strong expression of gta3, a ventral marker and cyt1, a novel type I cytokeratin expressed in the outer enveloping layer. Isolated 'embryonic shield', that corresponds to the amphibian organizer and amniote node, went on to express the mesodermal genes gsc and ntl, otx2, the anterior neural marker pax6, and posterior neural markers eng3 and krx20. The expression of these genes defined a precise anteroposterior axis in shield explants. When conjugated to animal caps, the shield frequently induced expression of anterior neural markers. More posterior markers were rarely induced, suggesting that anterior and posterior neural induction are separable events. Mesodermal genes were also seldom activated in animal caps by the shield, demonstrating that neural induction did not require co-induction of mesoderm in the caps. Strikingly, ventral marginal zone explants suppressed the low levels of otx2 in animal caps, indicating that ventral tissues may play an active role in axial patterning. These data suggest that anteroposterior patterning in the zebrafish is a multi-step process.

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

Primary mesenchyme cell migration in the sea urchin embryo: Distribution of directional cues

The directional migration of the primary mesenchyme cells (PMCs) of the sea urchin embryo is a critical step in the process of gastrulation. Although interactions between the migrating cells and the blastocoel environment are necessary for guiding the PMCs to their subequatorial target site, the nature of these interactions and the localization of guidance cues involved in directing the cells are not yet known. Previous studies have suggested that PMC migration is the result of random exploration and selective trapping at the target site by a pattern of adhesiveness in the ectoderm or basal lamina. To better characterize the distribution of guidance cues in the blastocoel we used a combination of time-lapse microscopy, microsurgery, and fluorescence photoablation to study the behavior of the migrating cells. By using fluorescence time-lapse microscopy, and a two-dimensional random-walk analysis of cell trajectories, we demonstrated that fluorescently labeled PMCs injected near the animal pole move in a directed fashion over a relatively long distance to reach the target site. This suggests that guidance cues are distributed globally throughout the embryo and are not restricted to the immediate ring area. To further test this hypothesis we investigated the migratory behavior of PMCs that were prevented from interacting directly with the target site. First, we examined the behavior of PMCs injected into animal embryo fragments lacking the target site. We found that PMCs move to the vegetal-most area of such embryo fragments, regardless of their size. Second, we studied the effects of photoablating a stripe of ectoderm between PMCs injected at the animal pole region (APR) and the target site. PMCs were found to accumulate along the ablated stripe and were unable to cross it for up to 6 hr after ablation. We also examined the migratory behavior of endogenous PMCs in embryos treated with lithium, a vegetalizing agent which shifts the position of the PMC ring toward the animal pole. We found that PMCs accumulated along an ablated stripe of ectoderm positioned below the shifted target site, suggesting that endogenous PMCs follow a set of directional cues to the target site which may be similar to those used by PMCs injected into the APR. As a whole, these results suggest that migrating PMCs follow a set of directional cues that are widely distributed throughout the blastocoel and that may be arranged in a gradient.

Fertilization of the lamprey (Lampetra japonica) eggs: Implication of the presence of fast and permanent blocks against polyspermy

AbstractWe have carried out a morphological examination to analyze the mechanism of monospermic fertilization in the lamprey, Lampetra japonica. Upon insemination, many sperm passed through a “tuft” of jelly that is found only on the animal pole area of the egg and underwent the acrosome reaction after they came into contact with the outer surface of the two‐layered vitelline envelope. The acrosomal filaments were admitted into the envelope and their tips touched the ooplasmic surface. The Hoechst dye transfer method revealed that a single sperm showed intense fluorescence before its nucleus penetrated the vitelline envelope; this indicated the actual fusion of the gametes. No other sperm with acrosomal filaments fluoresced, however. These findings indicate that a fast block against polyspermy is established at the plasma membrane level and prevents additional sperm‐egg fusion.The separation of the ooplasmic surface from the vitelline envelope (perivitelline space formation) was initiated beneath the margin of the animal pole region and progressed toward the animal and vegetal poles of the egg. At this stage, the head of a fertilizing sperm passed through the thickness of the vitelline envelope. The nuclei of supernumerary sperm were unable to enter the inner layer of the vitelline envelope. During the early phase of the perivitelline space formation at the animal pole region, many thin threads and one, or a small number, of the thick projection(s) of the ooplasm connected the envelope with the vitellus. A fertilizing sperm invariably moved along one of the thin threads and was embedded in the ooplasm at the animal pole region. Cytochalasin B inhibited sperm from passing through the inner layer of the vitelline envelope. When ionophore‐activated eggs were inseminated, the tips of the acrosomal filaments were prevented from touching the ooplasmic surface by the intervening presence of the perivitelline space. None of these sperm passed through the vitelline envelope. These findings suggest that the fusion of the acrosomal filament with the egg plasma membrane is a prerequisite for the passage of sperm through the inner layer of the vitelline envelope. Furthermore, actin‐mediated ooplasmic activity is probably involved in the passage. We conclude that the formation of the perivitelline space at the animal pole region contributes to the establishment of a permanent block against polyspermy in the lamprey egg. © 1994 Wiley‐Liss, Inc.

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 union 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.

Regulation of Primary Erythropoiesis in the Ventral Mesoderm of Xenopus Gastrula Embryo: Evidence for the Expression of a Stimulatory Factor(s) in Animal Pole Tissue

We have previously shown that the animal pole tissue from a st.10+ early gastrula Xenopus embryo stimulates the primary differentiation of erythrocytes in the ventral mesoderm in combination culture. To characterize the nature of this stimulation, various sizes and different portions of animal pole tissue were combined with the ventral mesoderm explants. The erythrocyte differentiation in terms of globin expression depended on the size of the animal pole tissue that was combined with the ventral mesoderm. No difference was observed in the potency of stimulation between the ventral and dorsal halves of animal pole tissue. We also found that animal pole tissue from as late as st.7 failed to stimulate both mRNA and protein levels of globin in the explant. Histological studies of the combination explant with st.7 animal pole tissue showed that epidermis, vesicle structure, and blood-cell-like cells developed in the explant, but very few blood cells expressed globin molecules. However, the stimulation of erythroid differentiation was restored if total (20 ng) or poly(A) + (0.2 ng) RNA from st.10+ animal pole tissue was previously injected at the 2-cell stage and the resulting animal pole tissue at st.7 was combined with st.10+ ventral mesoderm. Erythroid differentiation was also restored by injection with 1 ng of Xenopus bone morphogenetic protein-4 (XBMP-4) RNA. The effect of an extremely small dose of poly(A) + RNA on erythroid differentiation suggests that ia addition to XBMP-4 there exist substances, expressed later than st.7 in the animal pole region, which can stimulate erythrocyte differentiation in the ventral mesoderm.