pinecone/core-2020-05-10-deduplication huggingface dataset:
Antibodies to the Type II TGFβ Receptor Block Cell Activation and Migration during Atrioventricular Cushion Transformation in the Heart

AbstractEpithelial–mesenchymal transformation is a critical event in the development of many organ systems including the heart. Descriptive studies have implicated a number of factors in mediating this transformation, including transforming growth factor β (TGFβ). We now report that disruption of a TGFβ signal transduction complex by antibodies directed against the Type II TGFβ receptor blocks both the endocardial cell activation and subsequent migration that constitute transformation in the chick atrioventricular (AV) cushion. The Type II receptor was localized to both endothelial and endocardial cells of the chick embryo. Incubation of AV cushion explants from Stage 14, 16, and 18 embryos with antibody resulted in a blockade of AV endocardial cell transformation by greater than 50% as measured by mesenchyme formation. Similarly, the appearance of procollagen Type I, a marker of endocardial cell transformation, was blocked. In addition, within 2 hr after the incubation of activated Stage 18 explants with Type II antibody the rate of migration of transformed cells was decreased by 50%. These data suggest that TGFβ acts directly on AV cushion endocardial cells to stimulate epithelial–mesenchymal transformation and that TGFβ mediates at least two distinct components of AV cushion transformation, activation and migration
TheXenopusGATA-4/5/6 Genes Are Associated with Cardiac Specification and Can Regulate Cardiac-Specific Transcription during Embryogenesis

AbstractThe GATA family of nuclear factors has been implicated in the regulation of cell type-specific transcription. We report the isolation of theXenopusGATA-4 and GATA-6 cDNA clones and characterize the expression patterns of the xGATA-4/5/6 genes. By comparing the sequence of the cDNAs with those previously reported from chick and mammalian sources, we conclude that each is conserved across vertebrate evolution as a distinct gene product. Each gene is expressed in differentiated adult heart and gut, but maintains distinct transcript patterns in various other adult tissues. During embryogenesis, each gene displays a similar overlapping distribution of transcripts localized throughout the developing cardiogenic region. The xGATA-4 gene can be detected in dorsal cardiac progenitor rudiments prior to migration. Axis disruption experiments were used to demonstrate that transcription of these genes is intimately associated with the specification of cardiac progenitors. Ectopic expression of each gene is specifically capable of activating during embryogenesis the transcription of the cardiac genes encoding actin and myosin heavy chain α. The data are consistent with a primary role for the GATA-4/5/6 genes in regulating heart development
A cAMP Regulated K+-Selective Channel from the Sea Urchin Sperm Plasma Membrane

AbstractIon channels are deeply involved in sperm physiology. In sea urchin sperm cyclic nucleotide levels increase during quimotaxis and in the acrosome reaction (AR). Although cyclic nucleotides are second messengers known to directly or indirectly modulate ion channels, it is not clear how they modulate sperm responses to the egg outer layer. Here, we describe a cAMP regulated K+-selective channel from sea urchin sperm plasma membranes fused into planar bilayers that may have a role during sea urchin sperm quimotaxis and/or the AR. Its single channel conductance in 100 mMKCl is 103 pS. In bi-ionic experiments, the channel displayed a K+/Na+permeability ratio (PK+/PNa+) of ∼5. Thus, in sea water its reversal potential would be ∼−13 mV and channel opening would depolarize spermatozoa. The channel has low open probability (Po= 0.8 ± 0.2% at 0 mV applied voltage) and weak voltage dependence. Channel activity is reversibly up-regulated by cAMP in thecisbilayer side, but not by cGMP. This modulation followed a single Langmuir isotherm with an apparentkdof 200 μM.At this concentration the channel open probability at 0 mV increased up to 11-fold. TEA+blocked the channel only from thetransside. Also Ba2+intransblocked the channel in a voltage-dependent manner
Biphasic Activation of Fyn Kinase upon Fertilization of the Sea Urchin Egg

AbstractFertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm–egg binding and inhibitor studies have shown that tyrosine kinase activity is required for many aspects of egg activation. The present study demonstrates the presence of p59c-fynkinase in the sea urchin egg and examines the effect of fertilization on the activity of this enzyme. Fertilization had little effect on Fyn kinase activity during the first 2 min after insemination; however, activity had increased approximately eightfold between 5 and 15 min postinsemination. This initial, rapid increase in kinase activity was followed by a period of slightly elevated kinase activity, which was two- to threefold higher than that in the unfertilized egg. Bindin, as well as various parthenogenic agents known to activate the calcium- and pH-mediated pathways of egg activation, failed to elicit any change in enzyme activity, indicating that activation of the kinase required sperm-induced egg activation. However, phorbol ester treatment did induce a slow increase in kinase activity within 30 to 60 min of administration. These findings indicate that the p59fynkinase is activated within minutes of fertilization and may play a role in the egg activation process
A Nuclear Protein Regulated during the Transition from Active to Quiescent Phenotype in Cultured Endothelial Cells

AbstractPigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with theFUSgene product, TLS (Translocated in Liposarcoma; Crozatet al.,1993; Rabbitset al.,1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis
Developmentally Regulated Expression ofHsp70-2and aHsp70-2/lacZTransgene during Spermatogenesis

AbstractGerm cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation ofHsp70-2,we used mRNA primer-extension, reverse transcriptase PCR (RT–PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5′-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression ofHsp70-2during spermatogenesis. Results with multiple lines of transgenic mice containing promoter–reporter transgenes with varying lengths ofHsp70-2sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required forHsp70-2/lacZexpression in spermatocytes. Histochemical detection of transgene β-galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution ofHsp70-2andHsp70-2/lacZmRNAs was determined by Northern blot,in situhybridization, and RT–PCR, and it was found that upregulation of expression of bothHsp70-2andHsp70-2/lacZwas specific to the meiotic phase of spermatogenesis
Novel Postfertilization Inward Ca2+Current in Ascidian Eggs Ensuring a Calcium Entry throughout Meiosis

AbstractThe conductance change after fertilization in the oocyte of the ascidianCiona intestinalishas been followed by the whole cell patch-clamp technique. Two new inward currents, which are absent in unfertilized eggs, are elicited by hyperpolarization from a holding potential of +20 mV, which is the resting potential soon after fertilization. These currents reach their maximum level during the first meiotic division cycle, and then decrease in intensity, becoming almost undetectable at the 2-cell stage. These currents are most easily seen at high concentrations of barium. At least one, and likely both, of these currents appears to be carried by Ca ions. One of the currents is blocked by low concentrations of gadolinium; the other one is blocked by higher concentrations, although gadolinium at these levels does not block fertilization and the associated early depolarizing jump of the eggs. Thus these currents are not carried by channels that mediate the fertilization current. However, gadolinium blocks normal transition to 2-cell stage and blocks current oscillations synchronous to free calcium oscillations that occur normally in eggs around meiosis II. The electrical signature of calcium-release activated currents, taken together with these findings, suggests that these inward currents ensure a calcium entry pathway throughout meiosis. A plausible function of these currents may be to refill the Ca stores that are depleted after fertilization and that are required to progress into mitotic cell division. This interpretation is reinforced by experiments on unfertilized eggs with intracellular Ca stores depleted by thapsigargin, where both the newly described currents are observed
An Accumulation of p34cdc2at the End of Mouse Oocyte Growth Correlates with the Acquisition of Meiotic Competence

AbstractGrowing incompetent mouse oocytes released from follicular cells are unable to spontaneously resume meiosisin vitro.To identify the reasons for meiotic incompetence in these cells, the levels of p34cdc2/cyclin B kinase and p42MAPKbetween incompetent and competent oocytes were compared. p34cdc2was present at very low levels in incompetent oocytes and accumulated abruptly at the time of meiotic competence acquisition. By contrast, cyclin B and p42MAPKwere present at similar concentrations in both types of oocytes. Okadaic acid induced centrosome phosphorylation and meiotic reinitiation in incompetent oocytes, without inducing an increase in p34cdc2concentration. However, the p34cdc2present in incompetent oocytes was activated and all events following germinal vesicle breakdown were induced up to the formation of a metaphase I spindle including p42MAPKactivation, sustained increase in p34cdc2kinase activity, and translational activation of a dormant mRNA. We suggest that a threshold level of p34cdc2has to be reached for meiotic reinitiation to be spontaneously triggered: competence is restricted at a point preceding MPF activation. Whatever the mechanism involved in this restriction point, i.e., subthreshold concentration of p34cdc2and/or lack of an activator or presence of an inhibitor, it is bypassed by okadaic acid. Downstream of this point meiosis progresses up to metaphase I, even though p34cdc2concentration remains low
Multiple Actions of Stem Cell Factor in Neural Crest Cell Differentiationin Vitro

AbstractThe neural crest is a transient tissue of the vertebrate embryo that gives rise to most primary sensory neurons and pigment cells in the adult organism, among other cell types and tissues. Many neural crest cells are pluripotent in the sense that their progeny can generate more than one phenotype. The presence of pluripotent neural crest cell-derived cells at sites of terminal differentiation suggests that location-specific cues from the embryonic environment, such as growth factors, are involved in directing their survival, proliferation, and cell type specification. We have therefore examined the influences of one pertinent growth factor, stem cell factor (SCF), on neural crest cell development byin vitrocolony assay in a serum-free culture medium. SCF showed three major effects. (1) SCF is trophic for early neural crest cells, that is, either pluripotent cells and/or their more mature progeny. This effect occurs only if SCF is present throughout the culture period, and it is not observed when a neurotrophin is present in addition to SCF. (2) More colonies contain sensory neuron precursors in the presence of SCF. This effect is neutralized by NGF and neurotrophin-3 (NT-3), but not by brain-derived neurotrophic factor (BDNF). (3) The combination of SCF and any neurotrophin tested (NGF, BDNF, NT-3) is trophic for melanogenic cells, whereas SCF alone does not detectably affect melanogenesis. This suggests either that both types of factor are required for melanotrophic action or that melanogenic cells become dependent on neurotrophins after exposure to SCF. Our observation that SCF is required during the first half of the culture period only, and NGF during the second half only, indicates the latter possibility. Whereas coat color changes in the mouse mutantsW(c-kitdefect) andSteel(SCF defect) and severalin vivoandin vitrostudies by other investigators have shown previously that SCF is melanotrophic, they also indicated the requirement of an additional factor, or factors, in melanogenesis. Our data suggest that SCF affects neural crest cell development at multiple levels and that survival of melanogenic cells is mediated by a combination of SCF and a neurotrophin, rather than by SCF alone
The Role of FGF-3 in Early Inner Ear Development: An Analysis in Normal andkreislerMutant Mice

AbstractThe development of the otic placode is believed to depend on an inductive signal from the adjacent hindbrain. A candidate for this signal is FGF-3 (Int-2), which is expressed in the hindbrain adjacent to the future ear in rhombomeres 5 and 6 (r5 and r6). However,in vitrotests (Represaet al.(1991),Nature353, 561–563) conflict with findings from FGF-3 knockout mice (Mansouret al.(1993),Development117, 13–28). The former suggest that FGF-3 from the hindbrain is required to induce formation of the otocyst, while the latter imply that FGF-3 is required only in the later process of otocyst differentiation. We find that in normal embryos at early stages the gene is expressed not only in r5 and r6, but also in most of the hindbrain anterior to this and in the head ectoderm in the prospective otic placode region. Inkreislermutant embryos, however, there is no heightened expression in r5 and r6, but the early patch of expression in the prospective otic placode ectoderm is still seen and the otic vesicle still forms at nearly the normal place. Subsequent malformations of the inner ear inkreislerand in FGF-3 knockout mice are similar, involving failure of the development of the endolymphatic appendage. These findings argue that FGF-3 is not required as an inductive signal for invagination of the otic placode to form a vesicle, whose future site is already marked out independently of any localized FGF-3 signal from r5 and r6. FGF-3 does, however, appear to be required for a correct pattern of differentiation within the vesicle
Mechanism of an Evolutionary Change in Muscle Cell Differentiation in Ascidians with Different Modes of Development

AbstractWe have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidiansMolgula oculataandMolgula occultaare closely related species with different modes of development.M. oculataembryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereasM. occultaembryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin geneMocuMA1was isolated from anM. oculatagenomic library.MocuMA1is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5′ upstream region ofMocuMA1is sufficient to drive expression of alacZfusion construct in the larval muscle cells, implying that it is a functional gene.MocuMA1mRNA first appears in the prospective muscle cells ofM. oculataembryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected duringM. occultaembryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizingM. occultaeggs withM. oculatasperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting thattrans-acting factors responsible for muscle actin gene expression are conserved inM. occulta.The presence of thesetrans-acting factors was confirmed by showing that theMocuMA1/lacZfusion construct is expressed in the vestigial muscle cells ofM. occultalarvae. The orthologous larval muscle actin genesMoccMA1aandMoccMA1bwere isolated from aM. occultagenomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5′ upstream regions of theM. occultamuscle actin genes also contain numerous changes, expression ofMoccMA1a/lacZandMoccMA1b/lacZfusion constructs showed that they both retain specific promoter activity, although it is reduced inMoccMA1b.The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in thetrans-acting regulatory factors required for their expression
Purkinje Cell Lineage and the Topographic Organization of the Cerebellar Cortex: A View from X Inactivation Mosaics

AbstractWe utilized a strain of mice, derived from a radiation mutagenesis experiment and carrying an activity-attenuated allele of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD), to analyze the development of the cell lineage leading to cerebellar Purkinje neurons. Due to random X inactivation during early embryonic development, X-linked genes can be used to distinguish between clonally related populations of cells in X inactivation mosaics. Following histochemical staining for G6PD activity, the numeric proportions of Purkinje cells expressing either the wild-type or the mutant enzyme and the spatial distribution of these cellular phenotypes and their relation to anatomically and genetically defined cerebellar compartments were analyzed. Our data suggest that cerebellar Purkinje neurons originate from a limited pool of some 129 precursors. The size of this pool is different from the one derived from chimeric mice, allowing us to deduce the relative timing of Purkinje cell lineage restriction. Our data also show that Purkinje neurons of distinct lineage are extensively intermingled within the cerebellar cortex. Together, these findings suggest both a role for cell–cell communication in the development of genetically defined cerebellar compartments and a temporal window during which such cellular interactions may take place
Myogenic Determination Factor Expression in the Developing Avian Limb Bud: An RT–PCR Analysis

AbstractThis study examines the time of appearance of myogenic determination factor (MDF) transcripts in developing chick limbs and other embryonic tissues using reverse transcription–polymerase chain reaction (RT–PCR). In this assay, PCR amplification profiles of cDNA samples from stage 15–26 limb buds are compared to those of E11 heart, a tissue operationally defined as containing background levels of MDF transcripts which are thought to be nonfunctional. Myf 5 and MRF 4 transcripts are detected in stage 15 forelimbs, which is the earliest stage limb in which myogenic precursors have been detected following their migration from the somite. In contrast, MyoD and myogenin transcripts do not appear until about 1.5 days later, closer to the time of cytological muscle differentiation. A survey of MyoD, myf 5, and MRF 4 transcripts in other embryonic tissues reveals that MyoD transcripts are distributed in a highly skeletal muscle-specific pattern. In contrast, myf 5 and MRF 4 mRNAs are detected at significant levels in embryonic tissues that do not contain muscle or muscle precursor cells such as stage 9–15 neural tubes and stage 9–12 lateral plates, while these transcripts are found at very low levels in E11 heart and liver. The RT–PCR results are compared to those fromin situhybridization experiments as well as from cell culture assays of the myogenic potential of early limb cells
Primary Sequence and Developmental Expression Pattern of mRNAs and Protein for an α1Subunit of the Sodium Pump Cloned from the Neural Plate ofXenopus laevis

AbstractExpression of a catalytic α subunit of the sodium pump was followed in earlyXenopusembryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for α1subunit isoforms were cloned from a neural plate stageXenopuslibrary and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for α1subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed α1transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, α1mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, α1mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations