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Opaque tissues provide a challenge for live imaging of Xenopus laevis development; a problem solved by in vivo time-lapse X-ray microtomography that is shown to provide a high-resolution three-dimensional view of structural changes and dynamics of gastrulation, and that is applied to identify and analyse new aspects of gastrulation in frog embryos. New dimensions in vertebrate embryo microtomography Xenopus laevis - the South African clawed frog - is an important model organism, and much of our understanding of the vertebrate embryology derives from this system. But the study of gastrulation, the stage at which the embryo has formed three layers arranged around a central cavity, has been hampered by the lack of high-quality live-imaging methods useable on intact Xenopus embryos, which are opaque at early stages. Ralf Hofmann, Jubin Kashef and colleagues have developed a non-invasive in vivo time-lapse phase-contrast X-ray microtomography technique that allows the observation of gastrulation. By analysing individual cell trajectories, collective tissue motion and the evolution of morphological features, the authors visualize known gastrulation movements and reveal the formation of a structure not reported on previously. This new '4D' technique should be applicable in the fields of genetics, molecular and developmental biology and medicine. An ambitious goal in biology is to understand the behaviour of cells during development by imaging--in vivo and with subcellular resolution--changes of the embryonic structure. Important morphogenetic movements occur throughout embryogenesis, but in particular during gastrulation when a series of dramatic, coordinated cell movements drives the reorganization of a simple ball or sheet of cells into a complex multi-layered organism.sup.1. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants.sup.2,3, in fixed embryos.sup.4, in vivo using fluorescence microscopy.sup.5,6 or microscopic magnetic resonance imaging.sup.7. None of these methods allows cell behaviours to be observed with micrometre-scale resolution throughout the optically opaque, living embryo over developmental time. Here we use non-invasive in vivo, time-lapse X-ray microtomography, based on single-distance phase contrast and combined with motion analysis, to examine the course of embryonic development. We demonstrate that this powerful four-dimensional imaging technique provides high-resolution views of gastrulation processes in wild-type X. laevis embryos, including vegetal endoderm rotation, archenteron formation, changes in the volumes of cavities within the porous interstitial tissue between archenteron and blastocoel, migration/confrontation of mesendoderm and closure of the blastopore. Differential flow analysis separates collective from relative cell motion to assign propulsion mechanisms. Moreover, digitally determined volume balances confirm that early archenteron inflation occurs through the uptake of external water. A transient ectodermal ridge, formed in association with the confrontation of ventral and head mesendoderm on the blastocoel roof, is identified. When combined with perturbation experiments to investigate molecular and biomechanical underpinnings of morphogenesis, our technique should help to advance our understanding of the fundamentals of development., Author(s): Julian Moosmann [sup.1] , Alexey Ershov [sup.1] [sup.2] , Venera Altapova [sup.3] , Tilo Baumbach [sup.1] [sup.3] , Maneeshi S. Prasad [sup.4] , Carole LaBonne [sup.4] , Xianghui Xiao [...]

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance. germ cell | microRNA | RNA localization | vegetal | development www.pnas.org/cgi/doi/ 10.1073/pnas.1004401107

Platelet-derived growth factor (PDGF) signaling is essential for processes involving cell motility and differentiation during embryonic development in a wide variety of organisms including the mouse, frog, zebrafish, and sea urchin. In early Xenopus laevis embryos, PDGF-AA provides guidance cues for the migration of anterior mesendoderm cells as they move across a fibronectin-rich extracellular matrix. The long form of PDGF-A includes a positively charged carboxyl-terminal retention motif that can interact with the extracellular matrix and heparan sulfate proteoglycans (HSPGs). In this study we demonstrate that PDGF-AA binds directly to fibronectin and that this association is greatly enhanced by heparin. The PDGF-AA-fibronectin binding occurs across a broad range of pHs (5.5-9), which is significant because the PDGF-guided migration of Xenopus mesendoderm cells occurs under basic extracellular conditions (pH 8.4). We further demonstrate that endogenous HSPG's are required for the PDGF-AA-guided mesendoderm movement, suggesting an in vivo role for HSPGs in mediating the interaction between PDGF-AA and fibronectin. embryo | embryonic development | mesendoderm | mesoderm doi/10.1073/pnas.0902510106

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis. amphibian | skin | cell death | T cell | remodeling doi/10.1073/pnas.0708837106

The System L transporter facilitates cellular import of large neutral amino acids (AAs) such as Leu, a potent activator of the intracellular target of rapamycin (TOR) pathway, which signals for cell growth. System L is an AA exchanger, proposed to accumulate certain AAs by coupling to dissipation of concentration gradient(s) of exchange substrates generated by secondary active AA transporters such as System A (SNAT2). We addressed the hypothesis that this type of coupling (termed tertiary active transport) acts as an indirect mechanism to extend the range of AA stimulating TOR to those transported by both Systems A and L (e.g., Gln) through downstream enhancement of Leu accumulation. System A overexpression enabled Xenopus oocytes to accumulate substrate AAs (notably Ser, Gln, Ala, Pro, Met; totaling 2.6 nmol/oocyte) from medium containing a physiological AA mixture at plasma concentrations. Net accumulation of System L (4F2hc x LAT1) substrates from this medium by System L-overexpressing oocytes was increased by 90% (from 0.7 to 1.35 nmol/oocyte; mainly Leu, Ile) when Systems A and L were coexpressed, coincident with a decline in accumulation of specific System A substrates (Gln, Set, Met), as expected if the latter were also System L substrates and functional coupling of the transport Systems occurred. AA flux coupling was confirmed as trans-stimulation of Leu influx in System L-expressing oocytes by Gin injection (0.5 nmol/oocyte). The observed changes in Leu accumulation are sufficient to activate the TOR pathway in oocytes, although intracellular AA metabolism limits the potential for AA accumulation by tertiary active transport in this system. nutrient transport; target of rapamycin pathway; glutamine; leucine; amino acid exchanger

Bicarbonate transporters are regulated by signaling molecules/ ions such as protein kinases, ATP, and [Ca.sup.2+]. While phospholipids such as [PIP.sub.2] can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% C[O.sub.2]/33 mM HC[O.sup.-.sub.3] solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (-[V.sub.p] = -60 mV) containing a low-[Na.sup.+], nominally HC[O.sup.-.sub.3]-free solution. The current was 80-90% smaller in the absence of [Na.sup.+], approximately 75% smaller in the presence of 200 [micro]M DIDS, and absent in macropatches from [H.sub.2]O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing [Mg.sup.2+] in the presence or absence of vanadate and [F.sup.-] to reduce general phosphatase activity. Applying 5 or 10 [micro]M [PIP.sub.2] (diC8) in the presence of HC[O.sup.-.sub.3] induced an inward current in 54% of macropatches from NBC-expressing, but not [H.sub.2]O-injected oocytes. [PIP.sub.2]-induced currents were HC[O.sup.-.sub.3]-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of [Na.sup.+], and 90% smaller in the presence of 200 [micro]M DIDS. The polycation neomycin (250-500 [micro]M) reduced the [PIP.sub.2]-induced inward current by 69%; spermine (100 [micro]M) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HC[O.sup.-.sub.3]-induced inward currents by as much as 85%. In summary, [PIP.sub.2] stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters. acid-base | bicarbonate | pH | phosphatase | phospholipid

Histone H2A.X is an H2A variant present in multicellular organisms that is specifically phosphorylated on the serine in the C-terminal consensus sequence, canonically 'SQEY,' in response to DNA damage. We have recently shown the significance of phosphorylation of the penultimate tyrosine for maintenance and processing of the DNA damage response in mammalian cells. Here, we report the identification of distinct H2A.X variants in the eggs and early embryos of the frog Xenopus laevis that contain a C-terminal SQEF, among other changes; we have denoted these proteins as 'H2A.X-F.' H2A.X-F is present only in late-staged oocytes, eggs, and premidblastula transition embryos and is not present in somatic cells. Similar unannotated isoforms were identified in other rapidly developing aquatic species, such as Xenopus tropicalis, goldfish, and zebrafish, and in Arabidopsis and chickpea. Furthermore, we demonstrate by mass spectrometry and phospho-specific antibodies that H2A.X-F is phosphorylated in the absence of exogenous DNA damage, in both actively dividing, unperturbed embryos and cell-free egg extract in the absence and presence of DNA damage and S-phase checkpoint conditions. We propose that this isoform may be involved in modulating the cellular response to the rapid early cell cycles in externally developing species. chromatin | histone | variant | DNA damage

We utilize a novel, transgenic cell-labeling system to assess the embryonic derivation of cartilages in the post-metamorphic skull of anuran amphibians. Many of these cartilages form de novo at metamorphosis and have no obvious precursors within the larval skeleton. Most adult cartilages are derived from mandibular- or hyoid-stream neural crest, either individually or in combination; branchial-stream neural crest makes a modest contribution. Each stream also contributes to at least one cartilage in the middle ear or external ear. Four cartilages are composite elements; each is derived from at least two distinct cell populations. Many boundaries between adjacent neural-crest territories are cryptic insofar as they do not coincide with anatomical boundaries. The system of adult cranial segmentation revealed by these fate-mapping results differs in important respects from both the segmentation of the ontogenetically earlier larval skull and the cranial segmentation in amniotes. Most striking is the rostral 'inversion' of neural-crest-derived cartilages in Xenopus, such that mandibular stream-derived elements are deployed caudal to those derived from the hyoid stream, which predominate anteriorly. This novel pattern of rostral segmentation may be a consequence of the complex, biphasic life history that is characteristic of most species of living amphibians, and especially anurans, in which cranial architecture is significantly reconfigured at metamorphosis. Neural-crest derivation of the vertebrate skull is not invariant; instead, embryonic derivation of individual components of the cranial skeleton may vary widely among species.

We identified a functional domain (XlePABP2-TRP) of Xenopus laevis embryonic type II poly(A)-binding protein (XlePABP2). The NMR structure of XlePABP2-TRP revealed that the protein is a homodimer formed by the antiparallel association of [beta]-strands from the single RNA recognition motif (RRM) domain of each subunit. In each subunit of the homodimer, the canonical RNA recognition site is occluded by a polyproline motif. Upon poly(A) binding, XlePABP2-TRP undergoes a dimer-monomer transition that removes the polyproline motif from the RNA recognition site and allows it to be replaced by the adenosine nucleotides of poly(A). Our results provide high-resolution structural information concerning type II PABPs and an example of a single RRM domain protein that transitions from a homodimer to a monomer upon RNA binding. These findings advance our understanding of RRM domain regulation, poly(A) recognition, and are relevant to understanding how type II PABPs function in mRNA processing and human disease. nPABP2 | RRM | NMR | dimerization | PABPN1

Apical bumetanide-sensitive [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related [Na.sup.+]-[Cl.sup.-] cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by [sup.86][Rb.sup.+] influx in Xenopus laevis oocytes was markedly reduced by methyl-[beta]-cyclodextrin (M[beta]CD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/M[beta]CD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport. thick ascending limb; lipid raft; Xenopus oocyte; cholesterol depletion

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein-dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

At metamorphosis the Xenopus laevis tadpole exocrine pancreas remodels in two stages. At the climax of metamorphosis thyroid hormone (TH) induces dedifferentiation of the entire exocrine pancreas to a progenitor state. The organ shrinks to 20% of its size, and [approximately equal to]40% of its cells die. The acinar cells lose their zymogen granules and [approximately equal to]75% of their RNA. The mRNAs that encode exocrine-specific proteins (including the transcription factor Ptf1a) undergo almost complete extinction at climax, whereas PDX-1, Notch-1, and Hes-1, genes implicated in differentiation of the progenitor cells, are activated. At the end of spontaneous metamorphosis when the endogenous TH has reached a low level, the pancreas begins to redifferentiate. Exogenous TH induces the dedifferentiation phase but not the redifferentation phase. The tadpole pancreas lacks the mature ductal system that is found in adult vertebrate pancreases, including the frog. Exocrine pancreases of transgenic tadpoles expressing a dominant negative form of the TH receptor controlled by the elastase promoter are resistant to TH. They do not shrink when subjected to TH. Their acinar cells do not dedifferentiate at climax, nor do they down-regulate exocrine-specific genes or activate Notch-1 and Hes-1. Even 2 months after metamorphosis these frogs have not developed a mature ductal system and the acinar cells are abnormally arranged. The TH-dependent dedifferentiation of the tadpole acinar cells at climax is a necessary step in the formation of a mature frog pancreas. dedifferentiation | thyroid hormone | Thyroid hormone receptor dominant negative | gene expression

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/DmrtlbY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by [approximately equal to] 3 kb of the 5'flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tad-poles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis. FISH | sex determination | transgenic | W-chromosome | ZZ/ZW

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes, L-Ascorbic acid transport was saturable (Ko.5 [approximately equal to] 70 [micro]M), temperature dependent ([Q.sub.l0] [approximately equal to] 5), and energized by the [Na.sup.+] electrochemical potential gradient. We obtained a [Na.sup.+]-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and [Na.sup.+] saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered [Na.sup.+], L-ascorbic acid, [Na.sup.+]. In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM [Na.sup.+], maximal charge translocation ([Q.sub.max]) was [approximately equal to] 25 nC, around a midpoint ([V.sub.o.5]) at -9 mV, and with apparent valence [approximately equal to] -1. [Q.sub.max] was conserved upon progressive removal of [Na.sup.+], whereas [V.sub.o.5] shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first [Na.sup.+] partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT 1 to vitamin C metabolism in health and disease. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte

Magnesium is an essential metal, but few selective transporters have been identified at the molecular level. Microarray analysis was used to identify two similar transcripts that are upregulated with low extracellular [Mg.sup.2+]. The corresponding cDNAs encode proteins of 131 and 123 amino acids with two predicted transmembrane domains. The two separate gene products comprise the family that we have termed 'membrane [Mg.sup.2+] transporters' (MMgTs), because the proteins reside in the membrane and mediate [Mg.sup.2+] transport. When expressed in Xenopus laevis oocytes, MMgT1 and MMgT2 mediate [Mg.sup.2+] transport as determined with two-electrode voltage-clamp analysis and fluorescence measurements. Transport is saturable [Mg.sup.2+] uptake with Michaelis constants of 1.47 [+ or -] 0.17 and 0.58 [+ or -] 0.07 mM, respectively. Real-time RT-PCR demonstrated that MMgT mRNAs are present in a wide variety of cells. Subcellular localization with immunohistochemistry determined that the MMgT1-hemagglutinin (HA) and MMgT2-V5 fusion proteins reside in the Golgi complex and post-Golgi vesicles, including the early endosomes in COS-7 cells transfected with the respective tagged constructs. Interestingly, MMgT1-HA and MMgT2-V5 were found in separate populations of post-Golgi vesicles. MMgT1 and MMgT2 mRNA increased by about threefold, respectively, in kidney epithelial cells cultured in lowmagnesium media relative to normal media and in the kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets. With the increase in transcripts, there was an apparent increase in MMgT1 and MMgT2 protein in the Golgi and post-Golgi vesicles. These experiments suggest that MMgT proteins may provide regulated pathways for [Mg.sup.2+] transport in the Golgi and post-Golgi organelles of epithelium-derived cells. micmarray analysis; two-electrode voltage clamp; fluorescence; Xenopus oocytes

CPEB, a cytoplasmic polyadenylation element-binding protein, plays an important role in translational control of maternal mRNAs in early animal development. During Xenopus oocyte maturation, CPEB undergoes a Cdc2-mediated phosphorylation- and ubiquitin-dependent degradation that is required for proper entry into meiosis II. However, the precise mechanism of CPEB degradation, including the identity of the responsible E3 ubiquitin ligase, is not known. Here, we show that the [SCF.sup.[beta]-TrCP] E3 ubiquitin ligase complex targets CPEB for degradation during Xenopus oocyte maturation. [beta]-TrCP, the F-box protein of [SCF.sup.[beta]-TrCP], specifically binds to a sequence [sub.190][TSGFSS.sub.195] (termed here the TSG motif) of CPEB, thereby targeting CPEB for degradation. [beta]-TrCP binding depends on phosphorylation of Thr-190, Ser-191, and Ser-195 in the TSG motif. Among these residues, Ser-191 is phosphorylated by the Polo-like kinase Plx1, which binds CPEB at a specific Thr-125 residue prephosphorylated by Cdc2. Finally, Cdc2-mediated phosphorylation of other multiple Ser residues, previously implicated in CPEB degradation, is required for both Thr-125 phosphorylation and [beta]-TrCP binding, presumably causing conformational changes of CPEB. We propose that Cdc2 and PIx1 sequentially phosphorylate CPEB and target it for [SCF.sup.[beta]-TrCP] -dependent degradation in Xenopus oocytes. We suggest that many other proteins carrying the TSG-like motif may be targeted by [SCF.sub.beta]-TrcP]. cdc2 | phosphorylation | Plk1/Plx1 | [SCF.sup.[beta]-TrCP] ubiquitin ligase | translation

Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oo-cytes, with similar [EC.sub.50] values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins. fluorescent tag | receptor expression | Xenopus oocytes | kainate | glutamate

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/ Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chkl in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin. cell cycle | checkpoint | cyclin | DNA damage

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) using buckminsterfullerene ([C.sub.60]) as the primary ion source has the ability to generate chemical images of surfaces with high sensitivities and minimal chemical damage. We studied the application of [C.sub.60.sup.+] to depth profile a biological cell surface in a controlled manner and to subsequently image the revealed subsurfaces, in order to generate three-dimensional molecular images of the biological system. Such an analytical tool not only enables the surface localization of molecular species to be mapped but also enables the biomolecular distribution as a function of depth to be investigated with minimal sample preparation/intervention. Here we demonstrate the technique with a freeze-dried Xenopus laevis oocyte, which is a single cell. A [C.sub.60.sup.+] ion beam was used with computer-controlled analyses and etch cycles. Mass spectra derived from the surface revealed peaks corresponding to cholesterol (m/z 369) and other lipids at m/z 540-570 and 800-1000, in the positive ion mode, and lipid fatty acid side chains (e.g., m/z 255) in the negative ion mode. To our knowledge, this is the first demonstration of the 3D biomolecular imaging within an actual biological system using TOF-SIMS.

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to > 100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, [Na.sup.+]-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification. expression studies; aquaporin 2 mutations

The mechanisms underlying regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial [Na.sup.+] channel (ENaC) in Xenopus oocytes are controversial. CFTR's first nucleotide binding domain (NBD-1) may be important in these interactions, because mutations within NBD-1 impair these functional interactions. We hypothesized that an abnormal CFTR containing a non-NBD-1 mutation and able to transport chloride would retain regulatory interactions with murine ENaC (mENaC). We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR's first intracellular loop. I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Nature 410: 94-97, 2001). cRNAs encoding [alpha][beta][gamma]-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes, mENaC and CFTR functional expression were assessed by two-electrode voltage clamp, mENaC whole oocyte expression was determined by immunoblotting, and surface expression was quantitated by surface biotinylation. Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/IBMX. Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride. cystic fibrosis transmembrane conductance regulator; genistein

The regulation of protrusive activity of deep neural cells by notoplate tissues in Xenopus laevis is discussed.

The effects of Xenopus Neurotrophin Receptor Homolog 1 on mesodermal gene expression and cell death are presented.

Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

Oncoprotein 18/Stathmin (Op18) is a microtubule-destabilizing protein that is inhibited by phosphorylation in response to many types of signals. During mitosis, phosphorylation of Op18 by cdc2 is necessary but not sufficient for Op18 inhibition. The presence of mitotic chromosomes is additionally required and involves phosphorylation of Set-16 in Xenopus Op18 (and/or Ser-63 in human). Given that Ser-16 is an excellent Aurora A (Aut-A) kinase consensus phosphorylation site and the Aurora kinase inhibitor ZM447439 (ZM) blocks phosphorylation in the activation loop of Aur-A, we asked whether either Aur-A or Aurora B (Aur-B) might regulate Op18. We find that ZM blocks the ability of mitotic chromatin to induce Op18 hyperphosphorylation in Xenopus egg extracts. Depletion of Aur-B, but not Aur-A, blocks hyperphosphorylation of Op18, and chromatin assembled in the absence of Aur-B fails to induce hyperphosphorylation. These results suggest that Aur-B, which concentrates at centromeres of metaphase chromosomes, contributes to localized regulation of Op18 during the process of spindle assembly. aurora kinase inhibitor | mitosis | mitotic spindle

Using a sudy on Xenopus embryos, the role of NF-protocadherin and its partner TAF1/Set in cell-cell interactions and adhesions in neural folds and hence in neurulation, is discussed.

With-no-lysine kinase-1 (WNK1) gene mutations cause familial hyperkalemic hypertension (FHHt), a Mendelian disorder of excessive renal [Na.sup.+] and [K.sup.+] retention. Through its catalytic activity, full-length kinase-sufficient WNK1 (L-WNK1) suppresses its paralog, WNK4, thereby upregulating thiazide-sensitive Na-Cl cotransporter (NCC) activity. The predominant renal WNK1 isoform, KS-WNK1, expressed exclusively and at high levels in distal nephron, is a shorter kinase-defective product; the function of KS-WNK1 must therefore be kinase independent. Here, we report a novel role for KS-WNK1 as a dominant-negative regulator of L-WNK1. [Na.sup.+] transport studies in Xenopus laevis oocytes demonstrate that KS-WNK1 downregulates NCC activity indirectly, by inhibiting L-WNK1. KS-WNK1 also associates with L-WNK1 in protein complexes in oocytes and attenuates L-WNK1 kinase activity in vitro. These observations suggest that KS-WNK1 plays an essential role in the renal molecular switch regulating [Na.sup.+] and [K.sup.+] balance; they provide insight into the kidney-specific phenotype of FHHt. distal nephron; thiazide-sensitive sodium-chloride cotransporter; with-no-lysine kinases; aldosterone

Bves/pop1a is a unique, highly conserved integral membrane protein expressed in embryonic epithelia and striated muscle. Although studies have proposed a role in epithelial morphogenesis, the function of Bves/pop1a in development is completely unknown. Here we show that Xenopus laevis Bves (Xbves) RNA and protein are expressed in epithelia of the early embryon. Transfection of Xbves into nonadherent mouse L cells confers cell/cell adhesion. Global inhibition of Xbves function by morpholino injection into two-cell embryos arrests development at gastrulation by deregulating the epithelial movements of epiboly and involution. Clonal inhibition of Xbves activity within the A1 blastomere and its derivatives completely randomizes movement of its progeny within otherwise normally differentiating embryos. These data demostrate that Bves/pop1a proteins play a critical role in epithelial morphogenesis and, specifically, in the cell movements essential for epithelial rearrangements that occur during X. laevis development. cell adhesion | gastrulation | embryo | convergent extension morpholino

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.

Coupling of functional [GABA.sub.B] receptors ([GABA.sub.B]R) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing [GABA.sub.B1a] receptor ([GB.sub.1a]R) fused to Cerulean, a brighter variant of cyan fluorescent protein, and [GABA.sub.B2] receptor ([GB.sub.2]R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that [GB.sub.1a]R-Cerulean and GB2R-Venus form a heterodimer. The [GABA.sub.B]R agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying [K.sup.+] currents in a concentration-dependent manner in oocytes expressing [GB.sub.1a]R and [GB.sub.2]R, or [GB.sub.1a]R-Cerulean and [GB.sub.2]R-Venus, together with G protein-activated inward-rectifying [K.sup.+] channels (GIRKs), but not in oocytes expressing [GB.sub.1a]R alone or [GB.sub.2]R alone together with GIRKs. Oocytes coexpressing [GB.sub.1a]R + [G[alpha].sub.i2]fused [GB.sub.2]R ([GB.sub.2]R-[G[alpha].sub.i2]) caused faster [K.sup.+] currents in response to baclofen. Furthermore, oocytes coexpressing [GB.sub.1a]R + GB2R fused to [[G.sub.[alpha].sub.qi5] (a chimeric [G[alpha].sub.q] protein that activates PLC pathways) caused PLC-mediated [Ca.sup.2+]-activated C1 currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing [GB.sub.1a]R-[G[alpha].sub.i2] or [GB.sub.1a]R-[G[alpha].sub.qi5] together with GB2R. BHK cells and Xenopus oocytes coexpressing [GB.sub.1a]R-Cerulean + a triplet tandem of [GB.sub.2]R-Venus-[G[alpha].sub.qi5] caused FRET and [Ca.sup.2+]-activated C1 currents, respectively, with a similar potency in BHK cells coexpressing [GB.sub.1a]R-Cerulean + [GB.sub.2]R-Venus and in oocytes coexpressing [GB.sub.1a]R + [GB.sub.2]R-[G[alpha].sub.qi5]. Our results indicate that functional [GABA.sub.B]R forms a heterodimer composed of [GB.sub.1]R and GB2R and that the signal transducing G proteins are directly coupled to [GB.sub.2]R but not to [GB.sub.1]R. fluorescence resonance energy transfer

Transport effectors of Ca(2+) are tightly modulated during Xenopus oocycte maturation; processes are described for maintaining elevated cytoplasmic Ca(2+) necessary for initiating embryonic development.

The role of commissural interneuron interactions in Xenopus vertebrate spinal cord are described, focusing on the point when axons turn longitudinally after crossing the midline and the resultant series of dorsal adjustments.

In many animals, including Drosophila, C. elegans, zebrafish and Xenopus, the germ line is specified by maternal determinants localised in a distinct cytoplasmic structure called the germ plasm. This is consists of dense granules, mitochondria, and specific localised RNAs. We have characterised the expression and properties of the protein encoded by Xpat, an RNA localised to the germ plasm of Xenopus. Immunofluorescence and immunoblotting showed that this novel protein is itself a major constituent of germ plasm throughout oogenesis and early development, although it is also present in other regions of oocytes and embryos, including their nuclei. We found that an Xpat-GFP fusion protein can localise correctly in cultured oocytes, in early oocytes to the 'mitochondrial cloud', from which germ plasm originates, and in later oocytes to the vegetal cortex. The localisation process was microtubule-dependent, while cortical anchoring required microfilaments. Xpat-GFP expressed in late stage oocytes assembled into circular fields of multi-particulate structures resembling endogenous fields of germ plasm islands. Furthermore these structures could be induced to form at ectopic sites by manipulation of culture conditions. Ectopic Xpat-GFP islands were able to recruit mitochondria, a major germ plasm component. These data suggest that Xpat protein has an important role in Xenopus germ plasm formation, positioning and maintenance. Keywords: Xenopus; Oocyte; Germ plasm; Germ line; Xpat; Microtubules; Mitochondrial cloud

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally ([[lambda].sub.max]) at ~360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM W-VII on the [[lambda].sub.max]-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity [Na.sup.+]/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is -70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a [K.sub.m] for succinate of 4 [micro]M, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site. SLC13 gene family; citric acid cycle intermediate; lithium

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDKl/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases. Keywords: Cell cycle; Cell-free extracts; Embryo; M-phase duration; Mitosis; Xenopus laevis

Proneural basic helix-loop-helix (bHLH) transcription factors are critical positive regulators of neuronal differentiation in a variety of species and are required for proper differentiation of various subtypes of neurons. Although bHLH factors demonstrate some unique functions during neural development, they share the ability to regulate neuronal differentiation, potentially by targeting overlapping sets of genes. To assess this, we performed a screen in ectoderm animal cap tissue to identify direct transcriptional targets shared by two Xenopus ato-related bHLH factors, Xath5 and XNeuroD. Candidate target genes identified in this screen include several transcriptional regulators (Xebf2, Xebf3, XETOR and NKL), an RNA binding protein (elrC), a cell cycle component (Xgadd45[gamma]) and several novel genes. Overexpression of either Xath5 or XNeuroD induced ectopic in vivo expression of these candidate target genes. Conversely, blocking ato-related bHLH activity prevented endogenous nervous system expression of these genes. Therefore, we have identified a set of genes that can be regulated by multiple ato-related bHLH factors and may function as critical effectors of proneural bHLH-mediated differentiation. Keywords: Proneural; bHLH transcription factor; Neuronal differentiation: Target genes

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP1), which is known to be a chaperone for the cote histones H2A and H2B in Drosophila and mammalian cells [Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480]. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly suggest that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs. Xenopus laevis | chromatosome assembly | cell-free system

The effect of C[O.sub.2]-induced acidification on transjunctional voltage ([V.sub.j]) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. C[O.sub.2] application greatly decreased the [V.sub.j] sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. C[O.sub.2] also affected the kinetics of [V.sub.j] dependent inactivation of junctional current ([I.sub.j]), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the C[O.sub.2] sensitivity of chemical gating such that even C[O.sub.2] concentrations as low as 2.5% significantly lowered junctional conductance (Gi). With Cx50 channels [G.sub.j] dropped by 78% with a drop in intracellular pH (p[H.sub.i]) to 6.83, whereas with Cx50-D3N channels [G.sub.j] dropped by 95% with a drop in p[H.sub.i] to just 7.19. We have previously hypothesized that the way in which [V.sub.i] gating reacts to C[O.sub.2] might be related to connexin's gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of C[O.sub.2] on [V.sub.j] gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels. Keywords: Cytostatic factor; Metaphase II arrest; Xenopus egg; Cyclin B degradation; The anaphase-promoting complex/cyclosome; Cdc20; Mos; MAP kinase

Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the Xenopus laevis tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers undergo programmed cell death. We conclude that muscle remodeling and contraction play key roles in the shortening process, Shortening is accompanied by a temporary 'heaping' of the epithelial cells into many layers at climax. Cells that face the lumen undergo apoptosis, By the end of metamorphosis, when the epithelium is folded into crypts and villi, the epithelium is a single-cell layer once again. Throughout this remodeling, DNA replication occurs uniformly throughout the epithelium, as do changes in gene expression. The larval epithelial cells as a whole, rather than a subpopulation of stem cells, are the progenitors of the adult epithelial cells. BrdUrd | caspase-3 | smooth muscle | epithelium | thyroid hormone

Bone morphogenetic protein (BMP) inhibition has been proposed as the primary determinant of neural cell fate in the developing Xenopus ectoderm. The evidence supporting this hypothesis comes from experiments in explanted 'animal cap' ectoderm and in intact embryos using BMP antagonists that are unregulated and active well before gastrulation. While informative, these experiments cannot answer questions regarding the tinting of signals and the behavior of cells in the more complex environment of the embryo. To examine the effects of BMP antagonism at defined times in intact embryos, we have generated a novel, two-component system for conditional BMP inhibition. We find that while blocking BMP signals induces ectopic neural tissue both in animal caps and in vivo, in intact embryos, it can only do so prior to late blastula stage (stage 9), well before the onset of gastrulation. Later inhibition does not induce neural identity, but does induce ectopic neural crest, suggesting that BMP antagonists play temporally distinct roles in establishing neural and neural crest identity. By combining BMP inhibition with fibroblast growth factor (FGF) activation, the neural inductive response in whole embryos is greatly enhanced and is no longer limited to pre-gastrula ectoderm. Thus, BMP inhibition during gastrulation is insufficient for neural induction in intact embryos, arguing against a BMP gradient as the sole determinant of ectodermal cell fate in the frog. Keywords: Gastrulation; Inhibition; Embryo

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa [Na.sup.+]-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the [P.sub.i]-dependent current. For examaple, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V [less than or equal to] -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport. KEY WORDS: mutagenesis site directed * electrophysiology * phosphate transport proteins * electrogenic * Xenopus laevis

The neural crest is a multipotent population of cells that arises at the neural plate border in the vertebrate embryo. We have previously shown that a member of the Sox family of transcription factors, Sox9, is a regulator of neural crest formation in Xenopus, as Sox9-depleted embryos failed to form neural crest progenitors. Here, we describe experiments that further investigate Sox9 function during neural crest development. Induction of neural crest progenitors in Xenopus is regulated by Wnt signaling. We show that this process is largely dependent on Sox9 function as Writ-mediated neural crest induction is inhibited in the context of Soxg-depleted embryos. Moreover, we demonstrate that Sox9 functions as a transcriptional activator during neural crest formation. Expression of a construct in which Sox9 DNA-binding domain (HMG box) is fused to the repressor domain of Drosophila engrailed blocked neural crest formation, thereby mimicking the phenotype of Sox9-depleted embryos. Finally, using a hormone-inducible inhibitory mutant of Sox9, lacking the transactivation domain, we show that Sox9 function is required for neural crest specification but not for its subsequent migration. Keywords: Neural crest; Specification; Migration; Sox9; Slug; Snail; Wnt; Xenopus

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by [H.sup.+] in the physiological range of pH. The apparent affinity for [H.sup.+] of ASIC1a and 1b is modulated by extracellular [Ca.sup.2+] through a competition between [Ca.sup.2+] and [H.sup.+]. Here we show that, in addition to modulating the apparent [H.sup.+] affinity, [Ca.sup.2+] blocks ASIC1a in the open state (I[C.sub.50] ~ 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (I[C.sub.50] > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by [Ca.sup.2+]. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial [Na.sup.+] channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by [Ca.sup.2+] of ASIC1a. showing that these two amino acids (E425 and A channels. D432) are crucial for [Ca.sup.2+] block. It has previously been suggested that relief of [Ca.sup.2+] block opens However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by [H.sup.+] and modulation of [H.sup.+] affinity by [Ca.sup.2+]. These results show that channel block by [Ca.sup.2+] mad [H.sup.+] gating are not intrinsically linked. KEY WORDS: epithelial [Na.sup.+] channel * ion channel * channel pore * Xenopus oocyte * channel gating