pinecone/core-2020-05-10-deduplication huggingface dataset:
Conditioned Medium from Activated Microglia Promotes Cholinergic Differentiation in the Basal Forebrainin Vitro

AbstractIn earlier studies we found that treatment with interferon-γ (IFN-γ) produced an 8- to 11-fold increase in choline acetyltransferase (ChAT) in cultured cells taken from Embryonic Day 16 (E16) septal nuclei with adjacent basal forebrain (SN/BF). Since younger cultures responded even more profoundly to IFN treatment, we have tested the possibility that the action of IFN (or its intermediate; see below) is to prompt the cholinergic differentiation of neuronal precursors. SN/BF cultures of various ages were labeled with a retrovirus engineered to express β-galactosidase (Lac-Z), and ChAT-positive descendants of the retrovirally labeled precursors were counted. IFN-γ treatment of cultures caused as much as an 8.8-fold increase in the proportion of ChAT-positive cells present in Lac-Z-positive clones, suggesting that IFN promoted cholinergic differentiation in precursor populations. By contrast, bFGF increased clone size but did not change the proportion of ChAT-positive cells. NGF affected neither. Only ameboid microglia present in the cultures responded to IFN with characteristic nuclear translocation of the signal transducing molecule p91, suggesting that a microglial-derived molecule may mediate the action of IFN. Consistent with this hypothesis, conditioned media from cultures of enriched, activated microglia also increased ChAT activity in a dose-dependent fashion. Conditioned media from an unstimulated macrophage/monocyte cell line (RAW 264.7) also proved extremely efficacious in raising ChAT activity. In addition, conditioned media from both activated microglia and RAW 264.7 cells increased the proportion of ChAT-positive cells in retrovirally labeled clones to the same extent as IFN itself, suggesting the possibility that they contain the molecule(s) that mediates the action of IFN. Preliminary characterization of this molecule suggests that it is a very stable and large protein. Together these data suggest that a molecule promoting cholinergic differentiation is produced by activated microglia and other macrophage-like cells. The identity of this molecule and its precise role in normal development await its further purification
Role of Notochord in Specification of Cardiac Left–Right Orientation in Zebrafish andXenopus

AbstractThe left–right body axis is coordinately aligned with the orthogonal dorsoventral and anterioposterior body axes. The developmental mechanisms that regulate axis coordination are unknown. Here it is shown that the cardiac left–right orientation in zebrafish (Danio rerio) is randomized in notochord-defectiveno tailandfloating headmutants.no tail(Brachyury) andfloating head(Xnot) encode putative transcription factors that are expressed in the organizer and notochord, structures which regulate dorsoventral and anterioposterior development in vertebrate embryos. Results from dorsal tissue extirpation and cardiac primordia explantation indicate that cardiac left–right orientation is dependent on dorsoanterior structures including the notochord and is specified during neural fold stages inXenopus laevis.Thus, the notochord coordinates the development of all three body axes in the vertebrate body plan
Genetic Analysis of theDrosophilaβ3-Tubulin Gene Demonstrates That the Microtubule Cytoskeleton in the Cells of the Visceral Mesoderm Is Required for Morphogenesis of the Midgut Endoderm

AbstractWe have investigated the cellular basis for lethality of mutant alleles of theDrosophila melanogasterβ3-tubulin gene,βTub60D.Lethal β3 mutations can be grouped into two classes: the most severe mutations (Class I alleles) cause death during the first larval instar, while weaker alleles (Class II) cause death in later larval stages or in early pupal development. Since β3 is not expressed during larval development, lethality of the Class I mutations must reflect essential functions of β3 in embryogenesis. β3-tubulin is zygotically expressed during midembryogenesis in the developing mesoderm, and the major site of β3 accumulation is in the developing muscles during myogenesis. We show that the embryonic pattern of β3 expression, including accumulation in the developing musculature, is conserved in otherDrosophilaspecies. However, we found that loss of β3 function does not cause discernible defects in either the ultrastructure or function of the larval muscle. Thus β3-tubulin is dispensable in its highest site of accumulation. Rather, the essential site of function of β3 in embryos is in cells of the visceral mesoderm. Lethality of Class I alleles is caused by defects in midgut morphogenesis and failure of gut function. Although the folding pattern is irregular and the gut is smaller than normal, a complete folded gut forms in mutant larvae, and the visceral muscle functions normally to move food through the gut. However, mutant larvae cannot absorb nutrients across the gut wall. Thus loss of β3 function in the mesoderm results in defects in the underlying endodermally derived layer of the gut. Our data provide an assay for cellular interactions between mesoderm and endodermal tissues and reveal a role for the microtubule cytoskeleton of the visceral mesodermal cells in differentiation of the endodermal cell layer of the larval gut
decapentaplegicOverexpression AffectsDrosophilaWing and Leg Imaginal Disc Development andwinglessExpression

AbstractWe have used the GAL4-UAS expression system to increase the level of expression of theDrosophilagenedecapentaplegic(dpp) in a pattern approximating its normal pattern in leg and wing imaginal discs. Intermediate increases ofdppexpression have little effect in wing discs but high levels ofdppoverexpression lead to reduction of the scutellum and duplication of posterior wing structures. In leg discs intermediate increases cause supernumerary outgrowths of ventral leg structures in the anterior–ventral region. Greater increases ofdppexpression cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. The defects observed in both legs and wings appear to arise through dose-dependent effects ofdpponwingless(wg) expression. A high level ofdppoverexpression in the wing disc causes reduction ofwgexpression in the presumptive scutellar region, consistent with the subsequent reduction of the scutellum. An intermediate increase ofdppexpression in leg discs induces the expansion ofwgexpression into the ventral outgrowths. At higherdppexpression levels, ventralwgexpression in leg discs is eliminated, consistent with the loss of ventral leg cuticle. In the leg disc end knob and in the wing margin primordium, wherewganddppcooperate in producing distal outgrowth,dppoverexpression has no detectable effect either on patterning or onwgexpression. We propose that a critical role fordppin other regions of the leg and wing discs is to reduce or block the expression ofwg.This role ofdppis supported by the observation that ectopicwgexpression is detected in imaginal discs wheredppsignaling is compromised by lowering the activity of one of its receptors,tkv.This antagonism betweendppandwgexpression may be critical to assigning only one disc region as the distal organizer
Extracellular cAMP Depletion Triggers Stalk Gene Expression inDictyostelium: Disparities in Developmental Timing and Dose Dependency Indicate That Prespore Induction and Stalk Repression by cAMP Are Mediated by Separate Signaling Pathways

AbstractDuringDictyosteliumdevelopment, amoebae differentiate into spores and stalk cells. Earlier studies showed that extracellular cAMP is essential for induction of prespore differentiation and that cAMP represses stalk gene expressionin vitro.We show that the repressive pathway is operativein vivo,because activation of the stalk-specific promoter region of theecmBgene is strongly enhanced by overexpression of a phosphodiesterase that depletes extracellular cAMP. To test whether a single cAMP transduction pathway controls the choice between prespore or stalk cell differentiation, we compared the timing and dose dependency of the effects of cAMP on both responses. Cells acquire competence for cAMP repression ofecmBpromoter activity 4 hr later than for prespore gene induction. Half-maximal prespore induction requires 30 μMstable cAMP analog Sp-cAMPs, whileecmBinduction is half-maximally repressed by 200 nMSp-cAMPs, which is equivalent to about 3 to 13 nMcAMP. At concentrations exceeding 10 μM,Sp-cAMPs stimulatesecmBexpression from the intact promoter, but not from the stalk-specific subregion. These data suggest that distinct signaling pathways operating at different developmental stages control induction of prespore genes on one hand and repression of stalk genes on the other. Both stalk gene repression and prespore gene induction by Sp-cAMPs are antagonized by millimolar adenosine concentrations. However, an adenosine analog that is resistant to extracellular metabolism is active at 10 μM.Since adenosine inhibits cAMP binding to cAMP receptors, it may facilitate stalk gene expression by reducing the perceived cAMP concentration
Intraretinal Grafting Reveals Growth Requirements and Guidance Cues for Optic Axons in the Developing Avian Retina

AbstractTo study environmental factors controlling the growth and navigation of optic axons in the eye, grafts of retinal, optic disc, optic tectum, and floor plate tissue were transplanted into organ-cultured embryonic chick or quail eyes. The growth of axons into and out of the graft was studied in cross sections of the cultured eyes and by DiI tracing in retinal whole mounts. Based on the location and trajectory of axons and based on the quantity of axons that entered and exited the grafts, several requirements for axonal navigation were established: (1) Axonal growth is restricted to an approximately 10-μm-thick layer at the vitreal surface of the retina. (2) The retinal neuroepithelium prior to axogenesis is nonpermissive for neurite outgrowth. This nonpermissive quality is transient and recedes peripherally as the differentiation of the retina progresses. (3) Embryonic axons are able to grow into neonatal and adult retinal grafts, demonstrating that older retina remains permissive for axonal growth. (4) The trajectory of axons into and from retinal grafts that had been rotated in their peripheral–central orientation showed that the retina has an inherent polarity that permits axon growth toward and away from the optic disc, but does not allow axon growth perpendicular to this direction. This centroperipheral cue operates locally rather than by long distance. (5) The optic disc provides an exit for the axons from the retina, but has no detectable neurotropic activity. Finally, optic axons from the host retina readily enter grafts of their target tissue, the optic tectum, but few axons are able to leave tectal transplants
External Mg2+Triggers Oscillations and a Subsequent Sustained Level of Intracellular Free Ca2+, Correlated with Changes in Membrane Conductance in the Oocyte of the PrawnPalaemon serratus

AbstractWe have provided evidence that external Mg2+induces correlated changes in [Ca2+]iand membrane current or potential in prawn oocytes without any requirement of fertilization, using the fluorescent Ca2+indicator Ca green dextran and voltage and current clamp methods. Replacement of Mg2+-free ASW with standard (40 mMMg2+)ASW triggered a diphasic [Ca2+]iresponse consisting of an oscillation period followed by a second state of sustained [Ca2+]ilevel devoid of oscillation, lasting about 70 min and up to 3 hr, respectively. In contrast, oocytes maintained for a long time in Mg2+-free ASW showed no changes in [Ca2+]iwhich remained at a basal concentration of about 0.2 μM.The simultaneous records of [Ca2+]iand membrane or current changes showed that the oscillation period started with a first [Ca2+]ipeak (about 1.7 μM) and was correlated with an initial transient membrane hyperpolarization or a transient initial outward current peak. The first [Ca2+]ipeak was followed by a slow decrease in [Ca2+]ifollowed by a series of [Ca2+]itransients, concurrent with a slow depolarization of the membrane or a related inwardly directed current. The oscillation period ended with an oscillatory plateau of [Ca2+]i(about 0.6 μM) lasting 49.6 ± 4.5 min, the onset of which was concomitant with a final membrane hyperpolarization or a related final outward current. A continuous contact between external Mg2+and the oocyte membrane was necessary to maintain the program of [Ca2+]iand membrane conductance changes. The sources of Ca2+mobilization are of internal origin for both the first peak and the subsequent series of [Ca2+]ioscillations, and are mainly of external origin for the oscillation plateau and for the second state of sustained [Ca2+]ilevel. The first and second step of the cortical reaction occurred during the oscillatory plateau and the second state of sustained [Ca2+]ilevel, respectively
Isolated Maize Zygotes Mimicin VivoEmbryonic Development and Express Microinjected Genes When Culturedin Vitro

AbstractWe established conditions for the regeneration of natural maize zygotes isolated from pollinated plants with the goal of investigating the molecular control of early embryogenesis in higher plants. Viable zygotes were excised from embryo sacs by minimal enzymatic digestion and microdissection. Viable zygotes transferred to coculture with androgenic microspores from barley developed into embryo-like structures in 61% of the cases. No development was observed when zygotes were cultured in the presence of maize anthers undergoing androgenetic embryogenesis. Zygote-derived embryo-like structures regenerated into fertile plants through secondary embryogenesis when transferred to solid medium. The first zygotic division was asymmetrical and bipolar structures similar to pretransitional embryos observedin plantawere later produced as observed using light and electron microscopy. Conditions for efficient microinjection of DNA into zygotes were established. Calcofluor and PATAG staining of zygotes showed that cell wall regeneration occurred as early as 20 min after enzymatic isolation and that after 2 hr, each zygote was bordered with cell wall material. Through quantitative microphotometry, DNA synthesis during the first cell cycle of the zygote was shown to occur between isolation and 12 hr of culture. Microinjection of two types of reporter genes (GUSgene and anthocyanin regulatory genes) demonstrates transient expression in plant zygotes. On average, 3.5% of microinjected zygotes showed transgenic expression. Reporter gene expression was observed in zygotes at different time points of their first cell cycle
Characterization of Changes in F-actin during Maturation of Starfish Oocytes

AbstractMajor morphological and mechanical changes occur in the starfish oocyte during maturation. Measurements made by quantitative fluorescence microscopy of fixed specimens stained with saturating levels of rhodamine-phalloidin demonstrated that changes in the level of F-actin in intact oocytes, in the endoplasm, and in the cortex may contribute to these changes. The level of F-actin increased transiently after exposure of oocytes to the maturation inducing hormone, 1-methyladenine (1-MA). This increase correlated with the formation of spikes on the cell surface. The level of F-actin decreased at the time of germinal vesicle breakdown (GVBD), which may account for the decrease in stiffness that occurs at this time. No increase in the level of F-actin was observed during formation of polar bodies, suggesting the existence of a secondary mechanism affecting oocyte stiffness. The changes in the amount of F-actin during oocyte maturation were largest in the cortex. The data also suggested that there are two distinct populations of cortical actin that are regulated both spatially and temporally; these are the F-actin in spikes and nonspike cortical F-actin. Changes in either or both of these populations of cortical actin were induced independently of GVBD by short exposures to 1-MA, induction of maturation with dithiothreitol, and pretreatment of immature oocytes with forskolin before adding 1-MA. Stabilization of F-actin by microinjection of phalloidin had no effect on GVBD. These results suggest that polymerization and depolymerization of actin during maturation are responsible for morphological and mechanical changes in the oocyte. In addition, the data suggest that the regulation of actin polymerization and depolymerization can be dissociated from GVBD
Crumbs, a Component of the Apical Membrane, Is Required for Zonula Adherens Formation in Primary Epithelia ofDrosophila

AbstractThe zonula adherens (ZA) is a cell–cell adherens junction that forms a belt in the apical most region of the lateral cell surface of many epithelia. It is composed of the cadherin–catenin complex and many associated proteins and is connected to a prominent belt of microfilaments. The ZA is believed to play an important role in the differentiation and behavior of epithelial tissues and thus contributes substantially to embryonic morphogenesis. InDrosophilaembryos the ZA is formed during and shortly after gastrulation from adherens junction material that appears on the cell surface during cellularization. A ZA is present in a subset of epithelia in theDrosophilaembryo called primary epithelia. A second specific marker for primary epithelia is the Crumbs protein, which in concert with the gene product ofstardustis required to maintain epithelial polarity. This report shows that both genes are required for the reorganization of adherens junction material into the ZA. Using immunoelectron microscopy it is shown that Crumbs is not a component of the ZA but is distributed over the entire apical cell surface and concentrated in the immediate vicinity of the ZA. These results indicate a rather direct requirement of an apical activity for the organization of the lateral membrane domain inDrosophilaprimary epithelia. It is proposed that the marginal zone of the apical cell surface contains acrumbs- andstardust-dependent retention mechanism for adherens junction material that aids in the formation of the ZA
DNA Sequences Mediating the Transcriptional Response of theMix.2Homeobox Gene to Mesoderm Induction

AbstractPeptide growth factors can initiate changes in cell fate inXenopusectodermal explants and induce the formation of mesoderm. Marker genes expressed in mesoderm allow the analysis of whether, or how much, induction has occurred, but do not tell us what molecules are involved in carrying out the response. In this report we describe the isolation of genomic and cDNA clones ofMix.2, a gene closely related to theXenopushomeobox geneMix.1, and demonstrate that the promoter of theMix.2 gene is responsive to mesoderm induction signals when linked to a CAT reporter and microinjected into developingXenopusembryos. Like the chromosomalMix.1 gene, microinjectedMix.2 gene plasmids respond to activin in the presence of cycloheximide in animal cap assays and also respond to the embryonic inductive signal in Nieuwkoop recombinants. The injected promoter does not respond to TGF-β2 or FGF. Deletion analysis of theMix.2 promoter demonstrated that sequences required for maximal transcriptional activity in response to mesoderm induction are scattered across a 290-bp region. This is the first report of a microinjected plasmid responding to immediate-early transcriptional activation in developingXenopusembryos. This assay reduces the complexity of the cellular response to embryonic induction to the simple question of which molecules activate theMix.2 promoter and provides a sensitive and rapid test with which to pursue the answer
Targeted Disruption ofhoxc-4Causes Esophageal Defects and Vertebral Transformations

AbstractMice carrying a nonfunctional allele ofhoxc-4have been generated by gene targeting. The phenotype of mice homozygous for this mutation is strikingly different from those reported in mice lacking the paralogous geneshoxa-4, hoxb-4,andhoxd-4.In contrast to the mutants of the paralogous family members,hoxc-4homozygotes do not manifest abnormalities in the cervical vertebrae, but instead show vertebral defects that extend from the second thoracic vertebra (t2) to t11. Therefore, defects do not correspond to the anterior limit of expression ofhoxc-4,but rather begin within the region of stronghoxc-4expression in the prevertebral anlagen (i.e., pv7–14). Whilehoxc-4mutant homozygotes that reach adulthood are fertile and appear outwardly normal, most die before weaning age. The high lethality appears to result from partial or complete blockage of the lumen of the esophagus over a large portion of its length, as well as disorganization of the esophageal musculature. Although theDrosophilahomolog ofhoxc-4, Deformed,is autoregulated, mutation of thehoxc-4gene does not affect transcription of its paralogous family members. However, inhoxc-4mutant embryos, transcription of both thehoxc-5andhoxc-6genes is altered. Employment of cis/trans analysis showed that thehoxc-4mutation acts in cis to affect the pattern ofhoxc-5expression. Therefore, this mutation is likely to cause a reduction ofhoxc-5function as well as complete loss ofhoxc-4function
Avian Serum Response Factor Expression Restricted Primarily to Muscle Cell Lineages Is Required for α-Actin Gene Transcription

AbstractSerum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating α-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal α-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal α-actin promoter–luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for α-actin gene transcription