pinecone/core-2020-05-10-deduplication huggingface dataset:
Paracrine Induction of Stem Cell Renewal by LIF-Deficient Cells: A New ES Cell Regulatory Pathway

AbstractThe propagation of pluripotential mouse embryonic stem (ES) cells is sustained by leukemia inhibitory factor (LIF) or related cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotencyin vivo.In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of thelifgene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathwayin vivocould play an important role in the regulation of pluripotency in the epiblast and account for the viability oflifr−/− andgp130−/− embryos
Response of “Naive” Cutaneous and Muscle Afferents to Potential Targetsin Vitro

AbstractIt is now well documented that motoneurons are specified to innervate particular target muscles prior to axon outgrowth. Here we investigate whether sensory neurons are similarly specified to innervate target skin or muscle, taking advantage of the avian trigeminal system where cutaneous and muscle afferents are anatomically separate. Using this system, we have previously shown that by embryonic day 10 (E10) (approximately 4–5 days after target innervation), regenerating cutaneous and muscle afferents differ in their response to various potential targetsin vitro,in manners consistent with their normal innervation patternsin vivo.Thus, by E10 these two populations of sensory neurons have distinct identities as skin and muscle afferents. In contrast, we report here that the responses of younger, naive cutaneous and muscle afferents that have not yet, or only recently, innervated peripheral targets are indistinguishable, regardless of the target tissue tested. These findings suggest that at stages when innervation is being established, cutaneous and muscle afferents, unlike motoneurons, may not yet have acquired rigidly specified identities and/or the ability to recognize and respond selectively to their appropriate peripheral targets
Programmed Cell Death duringXenopusDevelopment: A Spatio-temporal Analysis

AbstractProgrammed cell death (PCD) is an integral part of many developmental processes. In vertebrates little is yet known on the patterns of PCD and its role during the early phases of development, when embryonic tissue layers migrate and pattern formation takes place. We describe the spatio-temporal patterns of cell death during earlyXenopusdevelopment, from fertilization to the tadpole stage (stage 35/36). Cell death was analyzed by a whole-mountin situDNA end-labeling technique (the TUNEL protocol), as well as by serial sections of paraffin-embedded TUNEL-stained embryos. The first cell death was detected during gastrulation, and as development progressed followed highly dynamic and reproducible patterns, strongly suggesting it is an important component of development at these stages. The detection of PCD during neural induction, neural plate patterning, and later during the development of the nervous system highlights the role of PCD throughout neurogenesis. Additionally, high levels of cell death were detected in the developing tail and sensory organs. This is the first detailed description of PCD throughout early development of a vertebrate, and provides the basis for further studies on its role in the patterning and morphogenesis of the embryo
Stromal Cells Are Critical Targets in the Regulation of Mammary Ductal Morphogenesis by Parathyroid Hormone-Related Protein

AbstractParathyroid hormone-related protein (PTHrP) was originally identified as the tumor product responsible for humoral hypercalcemia of malignancy. It is now known that PTHrP is produced by many normal tissues in which it appears to play a role as a developmental regulatory molecule. PTHrP is a normal product of mammary epithelial cells, and recent experiments in our laboratory have demonstrated that overexpression or underexpression of PTHrP in the murine mammary gland leads to severe disruptions in its development. The nature of these phenotypes suggests that PTHrP acts to modulate branching growth during mammary development by regulating mammary stromal cell function. We now demonstrate that throughout mammary development, during periods of active ductal-branching morphogenesis, PTHrP is produced by epithelial cells, whereas the PTH/PTHrP receptor is expressed on stromal cells. In addition, we show that mammary stromal cells in culture contain specific binding sites for amino terminal PTHrP and respond with an increase in intracellular cAMP. Finally, we demonstrate that the mammary mesenchyme must express the PTH/PTHrP receptor in order to support mammary epithelial cell morphogenesis. These results demonstrate that PTHrP and the PTH/PTHrP receptor represent an epithelial/mesenchymal signaling circuit that is necessary for mammary morphogenesis and that stromal cells are a critical target for PTHrP's action in the mammary gland
Normal Differentiation of Cultured Lens Cells after Inhibition of Gap Junction-Mediated Intercellular Communication

AbstractThe cells of the vertebrate lens are linked to each other by gap junctions, clusters of intercellular channels that mediate the direct transfer of low-molecular-weight substances between the cytosols of adjoining cells. Although gap junctions are detectable in the unspecialized epithelial cells that comprise the anterior face of the organ, both their number and size are greatly increased in the secondary fiber cells that differentiate from them at the lens equator. In other organs, gap junctions have been shown to play an important role in tissue development and differentiation. It has been proposed, although not experimentally tested, that this may be true in the lens as well. To investigate the function of gap junctions in the development of the lens, we have examined the effect of the gap junction blocker 18β-glycyrrhetinic acid (βGA) on the differentiation of primary cultures (both dissociated cell-derived monolayers and central epithelium explants) of embryonic chick lens epithelial cells. We found that βGA greatly reduced gap junction-mediated intercellular transfer of Lucifer yellow and biocytin throughout the 8-day culture period. βGA did not, however, affect the differentiation of these cells into MP28-expressing secondary fibers. Furthermore, inhibition of gap junctions had no apparent effect on either of the two other types of intercellular (adherens and tight) junctions present in the lens. We conclude that the high level of gap junctional intercellular communication characteristic of the lens equatorin vivois not required for secondary fiber formation as assayed in culture. Up-regulation of gap junctions is therefore likely to be a consequence rather than a cause of lens fiber differentiation and may primarily play a role in lens physiology
Retinal Neurogenesis: The Formation of the Initial Central Patch of Postmitotic Cells

AbstractWe have investigated the relationship between the birthdate and the onset of differentiation of neurons in the embryonic zebrafish neural retina. Birthdates were established by a single injection of bromodeoxyuridine into embryos of closely spaced ages. Differentiation was revealed in the same embryos with a neuron-specific antibody, zn12. The first bromodeoxyuridine-negative (postmitotic) cells occupied the ganglion cell layer of ventronasal retina, where they formed a small cluster of 10 cells or less that included the first zn12-positive cells (neurons). New cells were recruited to both populations (bromodeoxyuridine-negative and zn12-positive) along the same front, similar to the unfolding of a fan, to produce a circular central patch of hundreds of cells in the ganglion cell layer about 9 h later. Thus the formation of this central patch, previously considered as the start of retinal neurogenesis, was actually a secondary event, with a developmental history of its own. The first neurons outside the ganglion cell layer also appeared in ventronasal retina, indicating that the ventronasal region was the site of initiation of all retinal neurogenesis. Within a column (a small cluster of neuroepithelial cells), postmitotic cells appeared first in the ganglion cell layer, then the inner nuclear layer, and then the outer nuclear layer, so cell birthday and cell fate were correlated within a column. The terminal mitoses occurred in three bursts separated by two 10-h intervals during which proliferation continued without terminal mitoses
Functional Interactions of Genes Mediating Convergent Extension,knypekandtrilobite,during the Partitioning of the Eye Primordium in Zebrafish

AbstractVertebrate eye development in the anterior region of the neural plate involves a series of inductive interactions dependent on the underlying prechordal plate and signals from the midline of the neural plate, including Hedgehog. The mechanisms controlling the spatiotemporal expression pattern ofhedgehoggenes are currently not understood. Cyclopia is observed intrilobite(tri) andknypek(kny) mutants with affected convergent extension of the embryonic axis during gastrulation. Here, we demonstrate thattrimutants show a high frequency of partial or complete cyclopia,knymutants exhibit cyclopia infrequently, whileknym119trim209double-mutant embryos have dramatically reduced convergent extension and are completely cyclopic. We analyzed the relationships between the convergent extension defect, the expression ofhedgehogand prechordal plate genes, and the formation of cyclopia inknym119andtrim209mutants. Our results correlate the cyclopia phenotype with the abnormal location ofhh-expressing cells with respect to the optic primordium. We show that cyclopia in these mutants is not due to an incompetence oftriandknycells to respond to Hedgehog signaling. Rather, it is a consequence of exceeding a critical distance (>40–50 μm) betweenhedgehog-expressing cells and the prospective eye field. We hypothesize that at this distance, midline cells are not in an appropriate position to physically separate the eye field and that HH and other signals do not reach the appropriate target cells. Furthermore,triandknyhave overlapping functions in establishing proper alignment of the anterior neural plate and midline cells expressingshhandtwhhgenes when the partitioning of the eye primordium takes place
The βL Integrin Subunit Is Necessary for Gastrulation in Sea Urchin Embryos

AbstractIntegrins are a family of cell adhesion molecules reported to mediate cellular interactions essential for normal embryonic morphogenesis. Here we describe a β integrin subunit that is expressed during early embryogenesis in the sea urchin embryo and appears to be necessary for normal development. The deduced amino acid sequence of βL is similar to vertebrate β integrin subunits, but is most closely related to the sea urchin βG subunit. Northern blots show that βL is expressed at all stages with maximum expression beginning during gastrulation. Immunolocalization andin situRNA hybridization show that in blastulae βL is expressed in the blastoderm and by the ring of bottle cells in the vegetal plate during the initial phase of gastrulation. Presumptive secondary mesenchyme cells express high levels of βL throughout elongation of the archenteron and in the pluteus βL is expressed by blastocoelar cells, skeletal mesenchyme, and pigment cells. Antibodies and Fab fragments against βL block spreading of dissociated embryonic cells on RGD (arginine–glycine–aspartate)-containing substrates. Treating embryos with anti-βL antibodies blocks the initial phase of gastrulation and interferes with the organization of actin filaments. Prior to gastrulation, the antibodies cause thickening of the blastoderm and later in development defects in skeletal patterning result. Probing for antibody in treated embryos indicates that it penetrates the ectoderm to cells within the blastocoel and is actively endocytosed. We propose that βL forms receptors that bind to RGD-containing ligands and anchors actin filaments. These receptors appear to be essential in several aspects of morphogenesis
TheamnGene Product Is Required in Extraembryonic Tissues for the Generation of Middle Primitive Streak Derivatives

AbstractThe primitive streak is the defining feature of the gastrulating mouse embryo. Currently, little is known in the mouse about the mechanisms that mediate the assembly of the primitive streak or about the signaling pathways that specify the different types of mesoderm and endoderm generated from the streak. To gain insight into primitive streak assembly and function, we have conducted a detailed phenotypic characterization ofamnionless,a transgene-induced insertional mouse mutation that arrests embryonic development during gastrulation. Our histological and molecular analyses, when examined in the context of the mouse gastrula fate map, lead to the model that middle streak formation is specifically impaired in theamnionlessmutant. Significantly, these observations argue that the formation of the middle streak is mediated by a pathway that is genetically separable from those that direct the specification of the distal and proximal streak regions. Intriguingly, our findings from wt ES cell ↔amnionless−/−blastocyst chimeras indicate that this proposed separate pathway for middle streak formation is dependent onamnionlessgene functions in the visceral endoderm
Heterogeneity of Neural Progenitor Cells Revealed by Enhancers in the Nestin Gene

AbstractUsing transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS, while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory element. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations
The Products ofribbonandrawAre Necessary for Proper Cell Shape and Cellular Localization of Nonmuscle Myosin inDrosophila

AbstractMutations in the genesribandrawcause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by our observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced inribandrawmutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by eitherriborrawmutations. Furthermore, mutations ofzipper,the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected byribandraw,and many of the phenotypes are similar to those ofribandraw.Therefore, the products ofribandrawmay be required for proper myosin-driven contraction of the actin cytoskeleton
Role of Two Series of Ca2+Oscillations in Activation of Ascidian Eggs

AbstractChanges in [Ca2+]iare an essential factor regulating egg activation. Matured ascidian eggs are arrested at metaphase I, and two series of [Ca2+]itransients have been observed after fertilization: Ca2+waves just after fertilization (Series I) and [Ca2+]ioscillation between the first and second polar body extrusion (Series II). We investigated mechanisms involved in the elevation of [Ca2+]iand the role of the [Ca2+]itransients during egg activation inCiona savignyi.The monoclonal antibody 18A10 against IP3receptor type 1, which inhibits IP3-induced Ca2+release in hamster and mouse eggs, did not show substantial inhibitory effects on series I or egg deformation, whereas Series II and the first cell division were inhibited by the antibody. Ruthenium red, an inhibitor of ryanodine receptor-mediated Ca2+release, had no apparent effect of [Ca2+]itransients and other events related to the egg activation. Microinjection of IP3into unfertilized eggs induced [Ca2+]itransients similar to those seen in Series I, whereas injection of cyclic ADP ribose, an agonist of ryanodine receptors, rarely induced [Ca2+]itransient. Adenophostin B, a potent nonmetabolizable agonist of IP3receptors, induced [Ca2+]ioscillations which continued after first polar body extrusion, without separation to two series, and led to extrusion of first and second polar bodies. These results suggest that Series II is driven by the mouse type 1-like IP3receptor while Series I seems to be mediated by another type of IP3receptor. Injection of IP3only induced the first polar body extrusion and the egg was arrested at metaphase II even when a higher amount of IP3was injected. On the other hand, reinjection of IP3after the first polar body extrusion led to emission of the second polar body. Thus, Series I and II of [Ca2+]itransients are likely to be required for metaphase–anaphase transition in meiosis
Calcium/Calmodulin-Dependent Protein Kinase II and Calmodulin: Regulators of the Meiotic Spindle in Mouse Eggs

AbstractElevation of intracellular free calcium causes egg activation by initiating a cascade of interacting signaling pathways that, in unison, act to remodel the cytoplasmic compartment and the nuclear compartment of the egg. We show here that calcium/calmodulin-dependent protein kinase II (CaM kinase II) is tightly associated with the meiotic spindle and that 5 min after egg activation there is a transient, tight association of calmodulin (colocalized with CaM kinase II) on the meiotic spindle. These correlative observations caused us to test whether activation of CaM kinase II mediated the chromosomal transit into an anaphase configuration. We demonstrate that calcium and calmodulin, at physiological levels, along with ATP were capable of driving the spindle (with its associated CaM kinase II) into an anaphase configuration in a permeabilized egg system. The transit into anaphase was dependent on the presence of both calcium and calmodulin and occurred normally when they were present at a ratio of 4 to 1. Peptide and pharmacologic inhibitors of CaM kinase II blocked the transit into anaphase, both in the permeabilized egg system and in living eggs (inhibitors of protein kinase C did not block the transit into anaphase). Using a biochemical approach we confirm that CaM kinase II increases in activity 5 min after egg activation and that a second increase occurs 45 min after activation at the approximate time that the contractile ring of the second polar body is constricting. This corresponds to the approximate time when calmodulin and CaM kinase II colocalize at several points in the activated egg including the region containing midzone microtubules. CaM kinase II appears localized on midzone microtubules as soon as they form and may have a role in specifying the position of the contractile ring of the second polar body
The Direction of Neurite Growth in a Weak DC Electric Field Depends on the Substratum: Contributions of Adhesivity and Net Surface Charge

AbstractWe investigated the influence of the growth surface on the direction ofXenopusspinal neurite growth in the presence of a dc electric field of physiological magnitude. The direction of galvanotropism was determined by the substratum; neurites grew toward the negative electrode (cathode) on untreated Falcon tissue culture plastic or on laminin substrata, which are negatively charged, but neurites growing on polylysine, which is positively charged, turned toward the positive electrode (anode). Growth was oriented randomly on all substrata without an electric field. We tested the hypothesis that the charge of the growth surface was responsible for reversed galvanotropism on polylysine by growing neurons on tissue culture dishes with different net surface charges. Although neurites grew cathodally on both Plastek substrata, the frequency of anodal turning was greater on dishes with a net positive charge (Plastek C) than on those with a net negative charge (Plastek M). The charge of the growth surface therefore influenced the frequency of anodal galvanotropism but a reversal in surface charge was insufficient to reverse galvanotropism completely, possibly because of differences in the relative magnitude of the substratum charge densities. The influence of substratum adhesion on galvanotropism was considered by growing neurites on a range of polylysine concentrations. Growth cone to substratum adhesivity was measured using a blasting assay. Adhesivity and the frequency of anodal turning were graded over the range of polylysine concentrations (0 = 0.1 < 1 < 10 = 100 μg/ml). The direction of neurite growth in an electric field is therefore influenced by both substratum charge and growth cone-to-substratum adhesivity. These data are consistent with the idea that spatial or temporal variation in the expression of adhesion molecules in embryos may interact with naturally occurring electric fields to enhance growth cone pathfinding