pinecone/core-2020-05-10-deduplication huggingface dataset:
Impaired Lung Branching Morphogenesis in the Absence of Functional EGF Receptor

AbstractThe mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene bothin vivoand in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome
Intracellular Injections of a Soluble Sperm Factor Trigger Calcium Oscillations and Meiotic Maturation in Unfertilized Oocytes of a Marine Worm

AbstractHow sperm trigger activating calcium transients in eggs remains a central, unresolved question in fertilization biology. To determine if a soluble sperm factor can generate a fertilization-like calcium response in the absence of sperm-egg binding, aqueous extracts of sperm from the nemertean wormCerebratulus lacteuswere mixed with Ca2+-sensitive fluorescent dyes and injected into unfertilized, metaphase-I-arrested oocytes. Based on confocal imaging analyses, unfertilized oocytes that had been injected with sperm extract routinely produced oscillating Ca2+waves and resumed meiotic maturation in a manner that closely resembled normal fertilization. Calcium oscillations and maturation were typically lacking in control oocytes that had been (i) injected with buffer alone or with buffer containing added calcium, (ii) given external treatments of the sperm factor, or (iii) injected with extracts made from cells other than sperm. Boiling or protease treatment essentially abolished the potency of the sperm extract, and nonboiled extracts retained full activity in >10-kDa fractions, but not in <10-kDa fractions. Collectively, such findings suggest that the sperm ofC. lacteuspossess a soluble protein that can bypass oolemmal surface receptors to act within the ooplasm as a trigger of repetitive Ca2+waves and meiotic maturation. Results obtained in this study are discussed with respect to the minimum amount of extract needed for egg activation and whether the oscillogenic substance is sufficiently concentrated in a single sperm to play a biological role during fertilization
Development of Mechanisms Regulating Intracellular Ca2+Concentration in Cardiac Muscle Cells of Early Chick Embryos

AbstractThe development of mechanisms for the regulation of intracellular-free calcium ion concentration ([Ca2+]i) was investigated in precardiac mesodermal cells (PMC) and cardiac muscle cells (CMC) from early chick embryos by microfluorometry using a Ca2+-sensitive fluorescent probe, fura-2, and transmission electron microscopy. Microfluorometry indicated that two types of regulatory mechanisms, involving the dihydropyridine receptor (DHPR) and the ryanodine receptor (RYR), are present in CMC when the heartbeat begins at the 8–9 somite stages. Nifedipine completely suppressed the beating of hearts isolated from embryos on Days 1.5 and 2. Ryanodine had no effect on the beating of hearts isolated from embryos on Day 1.5, though it completely suppressed beating in hearts from Embryonic Day 2. Microfluorometry revealed that a change occured in the Ca2+-regulating mechanisms of CMC on Day 2. Transmission electron microscopy showed the appearance in CMC, also on Day 2, of peripheral couplings with feet structures, and SR adjacent to the Z-line of myofibrils. These findings suggest that the calcium-induced calcium-release (CICR) mechanism appears in the CMC of the chick on the second day of embryonic development
Glial-Restricted Precursors Are Derived from Multipotent Neuroepithelial Stem Cells

AbstractNeuroepithelial cells in the developing ventricular zone differentiate into neurons, astrocytes, and oligodendrocytes. It is not known, however, whether this differentiation occurs in a single step or is a pathway utilizing intermediate more restricted precursor cells. To characterize the generation of glial cells from multipotent stem cells we have cultured neuroepithelial (NEP) cells from E10.5 rat embryos. Cultured NEP cells do not express any glial differentiation markers when grown on fibronectin/laminin under nondifferentiation conditions. NEP cells, however, differentiate into A2B5 immunoreactive cells which can subsequently give rise to oligodendrocytes and astrocytes. Clonal analysis of NEP cells demonstrates that the A2B5 immunoreactive cells arise in clones that contain neurons and astrocytes, indicating that A2B5+cells arise from multipotent NEP precursor cells. A2B5+cells, maintained as undifferentiated cells over multiple passages, can subsequently give rise to both oligodendrocytes and astrocytes. A2B5+cells, however, do not generate neurons. Thus A2B5+cells represent a restricted progenitor cell population that differentiates from a multipotent NEP cell. Based on our results we propose that differentiation of the multipotential NEP cells to terminally differentiated glial cells occurs via intermediate restricted precursors
The Coupling of Cyclic GMP and Photopolarization ofPelvetiaZygotes

AbstractUnidirectional blue light directs the rhizoid–thallus axis in the apolar zygotes ofFucusandPelvetia.Here, it is shown that blue light (but not red light) increased cyclic GMP levels ofPelvetiazygotes by about a factor of 2. When the increase in cyclic GMP was blocked by a guanylyl cyclase inhibitor, photopolarization was also blocked. Bathing the cells in a permeant cyclic GMP analog, which should tend to collapse intracellular cyclic GMP gradients, reduced the degree of photopolarization. Growing the cells in the dark in a gradient of the analog caused the rhizoids to tend to form on the low concentration side. It appears that the stimulation of the blue light photoreceptors on the side nearer the light activates guanylyl cyclase and results in a transcytoplasmic cyclic GMP gradient that is necessary for polarization
Live Astrocytes Visualized by Green Fluorescent Protein in Transgenic Mice

AbstractGreen fluorescent protein (hGFP-S65T) was expressed in transgenic mice under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Tissues from two independent transgenic lines were characterized by Northern blot analysis and by confocal microscopy. The expression pattern in these two lines was identical in all tissues examined, and similar to that found previously with alacZtransgene driven by the same promoter. Bright fluorescence was observed in the cell bodies and processes of unfixed or fixed astrocytes, using both whole mount and brain slice preparations, from multiple areas of the central nervous system. However, in contrast to GFAP–lacZtransgenics, retinal Müller cells expressed the GFP transgene in response to degeneration of neighboring photoreceptors. These data indicate that the 2.2-kb hGFAP promoter contains sufficient regulatory elements to direct expression in Müller cells, and that GFP is a suitable reporter gene for use in living preparations of the mammalian nervous system. Such mice should prove useful for studies of dynamic changes in astrocyte morphology during development, and in response to physiological and pathological conditions
A Potential Role of R-cadherin in Striated Muscle Formation

AbstractWe have examined the murine embryonic expression pattern of the cell adhesion molecule R-cadherin in muscle, kidney, thymus, and lung. In developing muscle, R-cadherin was first seen at 10.5–11.5 days postcoitum in the somitic myotome. Consistently, we found R-cadherin expressed at the highest levels in the myotome, early skeletal muscle, and smooth muscle (both vascular and visceral), while very low levels of R-cadherin were detected in the heart. The expression pattern and subcellular localization of R-cadherin in developing skeletal muscle indicate a possible role in myoblast cell–cell interactions during both primary and secondary myogenesis. In the developing kidney, R-cadherin was first detected at 10.5 days postcoitum in the mesonephric epithelial tubule cells. In the metanephric kidney, it was specifically expressed in the pretubular aggregates, comma- and S-shaped bodies, proximal tubules, and collecting ducts. Thus, in the kidney, R-cadherin was associated with the mesenchymal–epithelial transition. R-cadherin was also found in other developing epithelia, for example in the thymic epithelial cells. In the lung, R-cadherin was expressed at the highest levels in the smooth muscle surrounding the lung epithelial tubules. To test whether R-cadherin can direct formation of tissues, we constitutively expressed R-cadherin inE-cadherin-/-ES cells and examined histogenesis in teratomas derived from these cells. R-cadherin exclusively rescued formation of striated muscle and epithelia in the teratomas. R-cadherin's ability to form epitheliain vivowas substantiated by its ability to rescue formation of cystic embryoid bodiesin vitro.By comparing our data with the previously reported embryonic expression patterns and histogenetic activities of E- and N-cadherin, we suggest that R-cadherin plays an important role in the formation of striated muscle and possibly also of epithelia
Multiple PositivecisElements Regulate the Asymmetric Expression of theSpHEGene along the Sea Urchin Embryo Animal–Vegetal Axis

AbstractThe mechanism that establishes the maternally determined animal–vegetal axis of sea urchin embryos is unknown. We have analyzed thecis-regulatory elements of theSpHEgene ofStrongylocentrotus purpuratus,which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C.,Dev. Biol.171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negativeciselement is responsible for the lack of vegetalSpHEtranscription. Surprisingly, even short segments of the regulatory region containing only several identifiedciselements also direct nonvegetal expression. Furthermore, theSpHEbasal promoter functions effectively in vegetal cells in combination withcis-acting elements derived from the PMC-specific gene,SM50.We conclude that the spatial pattern ofSpHEtranscription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression ofSM50also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H.,Development121, 1957–1970, 1995). A chimeric promoter containing bothSpHEandSM50regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which theSpHEandSM50genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo
Genetic Interactions in Zebrafish Midline Development

AbstractMutational analyses have shown that the genesno tail(ntl, Brachyuryhomolog),floating head(flh,aNothomeobox gene), andcyclops(cyc) play direct and essential roles in the development of midline structures in the zebrafish. In bothntlandflhmutants a notochord does not develop, and incycmutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent incyc;flhdouble mutants than in eithercycorflhsingle mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects inflh;ntldouble mutants are not additive, but are most often similar to those inntlsingle mutants. This finding reveals that loss ofntlfunction can suppress phenotypic defects due to mutation atflh,and we interpret it to mean that the wild-type allele ofntl(ntl+) functions upstream toflhin a regulatory hierarchy. Loss of function ofntlalso strongly suppresses the floor plate deficiency incycmutants, for we found trunk floor plate to be present incyc;ntldouble mutants. From these findings we propose thatntl+plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development
Reorganization of the Cortical Actin Cytoskeleton during Maturation Division in theTubifexEgg: Possible Involvement of Protein Kinase C

AbstractTubifexeggs undergo a drastic reorganization of the cortical actin cytoskeleton during metaphase of the second meiosis. At the end of the first meiosis, the egg cortex displays only scattered actin filaments and tiny dots of F-actin; during the following 90 min, cortical F-actin gradually increases in amount, becomes organized into foci that are interlinked by actin bundles, and generates a geodesic dome-like organization. In this study, we have characterized this reorganization of the cortical actin cytoskeleton. In living eggs injected with rhodamine–phalloidin at the beginning of the second meiosis, cortical actin assembly (i.e., formation of actin foci and bundles) proceeds normally, but labeled F-actin is not found to be included significantly in the formed cortical actin network, suggesting that the increase in cortical F-actin is not simply ascribable to the recruitment of preexisting actin filaments. Cortical actin assembly can be induced precociously not only by calcium ionophore A23187 but also by a phorbol ester PMA, an agonist of protein kinase C (PKC). Conversely, the formation of actin foci and bundles is inhibited by PKC antagonists, although cortical F-actin increases to some extent in the presence of these inhibitors. Similar inhibition of the cortical reorganization is elicited in eggs whose intracellular free calcium level ([Ca2+]i) has been clamped low by microinjection of a calcium chelator BAPTA. The treatment of BAPTA-injected eggs with PMA results in the formation of actin foci and bundles. An experiment with eggs injected with fluo-3 shows that [Ca2+]iincreases during metaphase of the second meiosis. These results suggest that the reorganization of cortical actin during metaphase of the second meiosis requires activation of PKC, which depends on increases in [Ca2+]i
Xenopus laevisSperm–Egg Adhesion Is Regulated by Modifications in the Sperm Receptor and the Egg Vitelline Envelope

AbstractThe biochemical and ultrastructural changes in the envelope of theXenopus laevisegg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M.,Methods Cell Biol.36, 231–247, 1991; Larabell, C. A., and Chandler, D. E.,J. Electron Microsc. Tech.17, 294–318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm–egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J.,J. Cell Biol.136, 1099–1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure–function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors duringX. laevisfertilization
Mapping Sperm Binding Domains on the Sea Urchin Egg Receptor for Sperm

AbstractThe receptor on the surface of the egg of the sea urchinStrongylocentrotus purpuratusthat mediates species-specific binding of sperm is a 350-kDa cell surface glycoprotein. Earlier studies established that a recombinant protein encompassing a major portion of the N-terminal half of the receptor inhibited fertilization when tested in a competitive fertilization bioassay. To identify in more detail the sites in this domain of the receptor that are involved in binding sperm, a series of deletion constructs were expressed as glutathioneS-transferase fusion proteins and tested for inhibitory activity in a fertilization bioassay. In addition, a novel assay for directly testing the sperm binding activity of these proteins was developed. In this assay we quantitated sperm binding to recombinant proteins representing various domains of the receptor immobilized on glutathione agarose beads. Using this new assay, two domains in the N-terminal half of the receptor were found to be involved in sperm binding. One of the peptide domains, composed of 247 amino acids, binds both the sperm ofS. purpuratusand the sperm of another genus of sea urchin,Lytechinus pictus.In contrast, binding to the second domain consisting of a 32-amino-acid residue peptide was found to be genus specific; no binding ofL. pictussperm was observed. A working model is proposed incorporating these findings with earlier studies on the function of the oligosaccharide chains of the receptor. In this model it is postulated that the sperm initially interact with the nonspecific binding domain on the polypeptide and the sulfated O-linked oligosaccharide chains of the receptor. This interaction is followed by binding to the more specific polypeptide binding site on the receptor. It is proposed that only subsequent to binding at this second site can gamete fusion occur
Evidence for Distinct Serine Protease Activities with a Potential Role in Processing the Sperm Protein Fertilin

AbstractThe guinea pig sperm protein fertilin (previously termed PH-30) plays an important role in sperm–egg fusion, and was the first recognized membrane-anchored metalloprotease/disintegrin protein. Fertilin is a heterodimeric glycoprotein which undergoes at least two distinct proteolytic processing steps. Fertilin α is processed first, in the testis, whereas fertilin β is processed separately during sperm maturation in the epididymis. The final processing of fertilin β occurs immediately adjacent to its predicted integrin ligand domain, and exposes an epitope recognized by a fusion blocking monoclonal antibody. Here, we demonstrate that one or more serine protease activities associated with testicular sperm can process fertilin βin vitroin a fashion that closely mimics the processing pattern observedin vivoduring epididymal sperm maturation. In contrast, several proteases that were added to testicular sperm did not mimic the pattern observedin vivo.These findings raise the intriguing possibility that a fertilin β converting protease(s) activein vivomay originate from sperm, instead of from the epididymal epithelium. Further, we show that fertilin α is most likely processed intracellularly in the secretory pathway based on three observations: (i) only processed fertilin α, but not the precursor pro-α can be cell-surface biotinylated; (ii) some processed fertilin α is sensitive to endoglycosidase H, suggesting cleavage occurs prior to the medial Golgi apparatus; (iii) a reanalysis of the N-terminus of processed fertilin α showed that the proteolytic cleavage site is next to four arginine residues, a consensus sequence for intracellular subtilysin type pro-protein convertases. The N-terminal sequence analysis further showed that processed fertilin α contains an intact membrane anchored disintegrin domain, and not a truncated disintegrin domain as reported previously (Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D. G., Primakoff, P., and White, J. M.,Nature356, 248–252, 1992). Proteolytic processing is thought to play an important role in regulating the function of fertilin, and the present study represents a first step toward a better understanding of protease activities involved in the maturation of fertilin, and potentially other sperm surface proteins