pinecone/core-2020-05-10-deduplication huggingface dataset:
The Expression and Stage-Specific Localization of Protein Kinase C Isotypes during Mouse Preimplantation Development

AbstractSignaling events mediate many processes that act during embryogenesis to initiate the program of early development. Within the cell many of these changes are mediated through the activation or inactivation of kinases and phosphatases. Protein kinase C (PKC) is one kinase that has been shown to be involved in at least two developmental transitions during early development, fertilization and embryonic compaction. PKC is a family of kinases whose various isotypes have differing requirements for activation of the kinase that include the availability of calcium, diacylglycerol, and negatively charged phospholipids. The presence of more than one isotype in an egg or blastomere of the embryo would provide the possibility that different isotypes mediate distinct signaling pathways in the cells. To address this possibility the different isotypes of PKC were examined at the mRNA and protein levels during preimplantation development in the mouse. Our results demonstrate that seven isotypes of PKC are present during preimplantation development in mouse, some are of maternal origin and others appear after fertilization. Two isotypes have a stage-dependent nuclear localization. In addition, within each blastomere PKC isotypes occupy different subcellular locations in a stage-dependent fashion
Metaphase I Arrest and Spontaneous Parthenogenetic Activation of Strain LTXBO Oocytes: Chimeric Reaggregated Ovaries Establish Primary Lesion in Oocytes

AbstractOocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF1) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions
Characterization of Brachyury-Downstream Notochord Genes in the Ciona intestinalis Embryo

AbstractThe notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519–1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, β4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos
Cellular Basis of the Dynamic Behavior of the Imaginal Thoracic Discs during Drosophila Metamorphosis

AbstractThe eversion, migration, spreading, and fusion of the thoracic imaginal discs during metamorphosis of Drosophila are described using timed whole-mount preparations and several molecular markers. The leading edge of the migrating disc epithelia consists of two groups of cells, stalk cells (S cells) and specialized imaginal cells (I cells), that both express the gene puckered. With this and other markers, opening of the stalk, eversion of the discs, migration of the leading edges, and fusion of the imaginal epithelia can be visualized in detail. Fusion is initiated by S cells that migrate over the larval epithelium and constitute a bridge between two imaginal epithelia. S cells are subsequently lost and imaginal fusion is mediated by the I cells that remain at the site of fusion. The possible cellular basis of this process is discussed. Fusion along the dorsal midline of the notum from the mesothoracic wing discs occurs earlier than that of the prothoracic and metathoracic discs, which remain in a lateral position. For a relatively long period (30 h) the mesothoracic epithelium becomes attached to the head and abdomen, causing a temporary local discontinuity of the order of segments. Later the pro- and metathoracic discs intercalate between head and mesothorax and between abdomen and mesothorax, respectively, to reestablish the normal order
Spatial and Temporal Expression of a Polysphondylium Spore-Specific Gene

AbstractIn the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions −246 and −352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process
Cells Remain Competent to Respond to Mesoderm-Inducing Signals Present during Gastrulation in Xenopus laevis

AbstractDuring gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals
A Mode of Regulation of β-Catenin Signaling Activity in Xenopus Embryos Independent of Its Levels

AbstractThe signaling activity of β-catenin is thought to be regulated by phosphorylation of a cluster of N-terminal serines, putative sites for GSK3β. In the prevailing model in the literature, GSK3β-dependent phosphorylation of these sites targets β-catenin for ubiquitin-mediated degradation. Wnt signaling inhibits GSK3β activity and this blocks degradation, allowing β-catenin to accumulate and signal. We show here that β-catenin activity is not regulated solely by protein stability. Mutations in the putative GSK3β phosphorylation sites of β-catenin enhance its signaling activity, but this cannot be accounted for by accumulation of either total or cadherin-free protein. Instead, the mutant protein has a threefold higher specific activity than the wild type both in vivo and in an in vitro signaling assay. We conclude that the N-terminal serines convey a layer of regulation upon β-catenin signaling in addition to the effects these sites exert upon protein stability
Local Inhibition of Cortical Rotation in Xenopus Eggs by an Anti-KRP Antibody

AbstractThe dorsal–ventral axis of amphibian embryos is specified by the “cortical rotation,” a translocation of the egg cortex relative to the vegetal yolk mass. The mechanism of cortical rotation is not understood but is thought to involve an array of aligned, commonly oriented microtubules. We have demonstrated an essential requirement for kinesin-related proteins (KRPs) in the cortical rotation by microinjection beneath the vegetal cortex of an antipeptide antibody recognising multiple Xenopus egg KRPs. Time-lapse videomicroscopy revealed a striking local inhibition of the cortical rotation around the injection site, indicating that KRP-mediated translocation of the cortex is generated by forces acting across the vegetal subcortical region. Anti-tubulin immunofluorescence showed that the antibody disrupted both formation and maintenance of the aligned microtubule array. Direct examination of rhodamine-labelled microtubules by confocal microscopy showed that the anti-KRP antibody provoked striking three-dimensional flailing movement of the subcortical microtubules. In contrast, microtubules in antibody-free regions undulated only within the plane of the cortex, a significant population exhibiting little or no net movement. These findings suggest that KRPs have a critical role during cortical rotation in tethering microtubules to the cortex and that they may not contribute significantly to the translocation force as previously thought
sem-4 Promotes Vulval Cell-Fate Determination in Caenorhabditis elegans through Regulation of lin-39 Hox

AbstractVulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953–1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals
Cyclopamine Inhibition of Sonic Hedgehog Signal Transduction Is Not Mediated through Effects on Cholesterol Transport

AbstractCyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopamine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopamine is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. Second, by comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholesterol transport, we show that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds that block cholesterol transport by affecting the vesicular trafficking of the Niemann–Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol homeostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consistent with this model, we find that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells
A Role for Cyclin A1 in the Activation of MPF and G2–M Transition during Meiosis of Male Germ Cells in Mice

AbstractCell-cycle transition at G2–M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2–-M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2–M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases
Different Isoforms of Fasciclin II Play Distinct Roles in the Guidance of Neuronal Migration during Insect Embryogenesis

AbstractDuring the formation of the enteric nervous system (ENS) of the moth Manduca sexta, identified populations of neurons and glial cells participate in precisely timed waves of migration. The cell adhesion receptor fasciclin II is expressed in the developing ENS and is required for normal migration. Previously, we identified two isoforms of Manduca fasciclin II (MFas II), a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) and a transmembrane isoform (TM-MFas II). Using RNA and antibody probes, we found that these two isoforms were expressed in cell type-specific patterns: GPI-MFas II was expressed by glial cells and newly generated neurons, while TM-MFas II was confined to differentiating neurons. The expression of each isoform also corresponded to the motile state of the different cell types: GPI-MFas II was detected on tightly adherent or slowly spreading cells, while TM-MFas II was expressed by actively migrating neurons and was localized to their most motile regions. Manipulations of each isoform in embryo culture showed that they played distinct roles: whereas GPI-MFas II acted strictly as an adhesion molecule, TM-MFas II promoted the motility of the EP cells as well as maintaining fasciculation with their pathways. These results indicate that precisely regulated patterns of isoform expression govern the functions of fasciclin II within the developing nervous system
Xlim-1 and LIM Domain Binding Protein 1 Cooperate with Various Transcription Factors in the Regulation of the goosecoid Promoter

AbstractThe homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc–luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST–homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation
Patterning the Early Zebrafish by the Opposing Actions of bozozok and vox/vent

AbstractFish and frog embryos are patterned along the dorsal–ventral axis during the gastrula stage by opposing gradients of Bmps and Bmp inhibitory proteins. Three transcriptional repressors with partially overlapping expression domains have been proposed to be important mediators of Bmp function in Xenopus. We find that two related factors are expressed in the early zebrafish embryo. Although these factors are considerably divergent from the related Xenopus genes, they are expressed in domains similar to those of their Xenopus relatives throughout embryogenesis. Both of the zebrafish genes, which we have named vox and vent, are potent ventralizing factors in both zebrafish and Xenopus embryos. Using mutants in the Bmp pathway, we find that there are Bmp-dependent and Bmp-independent domains of vox expression, whereas vent is mostly dependent upon Bmp signaling. We show that ectopic vox or vent negatively regulates expression of the early dorsal gene bozozok (boz) and that ectopic boz eliminates vox and vent expression. Moreover, the normal exclusion of vox and vent from the organizer region is lost in boz mutant embryos. Our results show that boz and vox/vent are mutually antagonistic and indicate that the early establishment of the size of the organizer domain is dependent on an interplay between these early expressed transcriptional repressors