pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of Regulatory Regions Driving the Expression of the Drosophila spalt Complex at Different Developmental Stages

AbstractThe zinc finger transcription factors Spalt and Spalt-related have been implicated in multiple developmental processes. In the wing they are regulated by the secreted protein Decapentaplegic and participate in the positioning of the wing veins. The function of Spalt has been also analyzed during tracheal development and embryonic segmentation. Here, we present the isolation and characterization of novel spalt/spalt-related alleles, which analysis indicates that these genes cannot substitute for each other in the developmental processes studied. The mutants present embryonic or pupal lethality, with phenotypes consistent with the loss of spalt function. We also present a detailed functional analysis of the DNA regions implicated in the regulation of these genes. This regulation is complex, integrating the information from both negative and positive regulators, and it is modular, with discrete fragments of DNA directing expression to discrete regions in embryonic and larval tissues
Laminin and β1 Integrins Are Crucial for Normal Mammary Gland Development in the Mouse

AbstractWe have examined the role of integrin–extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell–matrix interactions in gland development, pellets containing function-perturbing anti-β1 integrin, anti-α6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking β1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of β1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-α6 integrin antibody, suggesting that α6 is not the important partner for β1 in this system. The studies with β1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)–induced tubulogenesis is dependent on functional β1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and β1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development
Oocyte Regulation of Kit Ligand Expression in Mouse Ovarian Follicles

AbstractKit ligand (KL), a product of granulosa cells in ovarian follicles, is a putative regulator of oocyte development. However, the factors that regulate KL mRNA levels in granulosa cells remain unclear. This study tested the hypothesis that oocytes regulate granulosa cell steady-state KL mRNA expression levels and that the characteristics of this regulation are dependent on the stage of growth and development of both oocytes and follicles. Levels of mRNA for the KL splice variants (KL-1 and KL-2) were shown to be high in granulosa cells from preantral follicles and then decline after follicular antrum formation. Preovulatory follicular development was associated with a dramatic increase in steady-state levels of KL-1 mRNA in mural granulosa but not cumulus cells. Regulation of these changes was examined in vitro using partly grown oocytes isolated from preantral follicles and fully grown oocytes isolated from preovulatory follicles. FSH increased the steady-state KL mRNA levels in preantral granulosa cells in vitro. Partly grown oocytes either increased or decreased KL mRNA levels in preantral granulosa cells depending on the absence or presence of FSH stimulation, respectively. Fully grown oocytes reduced the KL mRNA level in preantral granulosa cells and increased the ratio of KL-1 to KL-2 mRNA. In mural granulosa cell culture, FSH augmented testosterone-dependent elevation of the steady-state KL mRNA level, but had no effect alone. Fully grown oocytes reduced KL-2 but not KL-1 mRNA levels in mural granulosa cells treated with testosterone plus FSH, whereas fully grown oocytes reduced levels of both KL transcripts in cumulus cell culture. These effects of oocytes on steady-state KL mRNA expression levels in vitro explain the changes in granulosa cell KL mRNA levels observed during follicle development in vivo. The results therefore support the hypothesis that oocytes regulate granulosa cell kit ligand mRNA levels in a way that is characteristic of the stage of growth and development of the oocyte. Moreover, the results suggest that oocytes play a major role in promoting dynamic changes in gene expression by granulosa cells appropriate to the stage of follicular development
Early Specification of Oligodendrocytes in the Chick Embryonic Brain

AbstractOligodendrocytes are the myelin-forming cells in the central nervous system of vertebrates. In the rodent embryo, these cells have been shown to emerge from restricted territories of the neuroepithelium. However, a comprehensive view of the development of oligodendroglial populations from their ventricular sources remains to be established. As a first step toward this aim, we have examined in vivo the spatiotemporal emergence of oligodendrocytes in the chick embryonic brain. We have detailed the patterns of expression of three early markers of the oligodendroglial lineage: the plp/dm-20 and PDGFRα transcripts and the O4-reactive antigen. During embryonic development, these molecules showed a similar segmental pattern of expression. However, plp/dm-20+ cells were already observed, in the ventricular layer, at E2.5, i.e., 2 days before the appearance of O4+ and PDGFRα+ cells, suggesting that oligodendrocyte precursors arise nearly simultaneously with neurons. In the chick embryonic brain, the onset of expression of plp/dm-20 appears therefore to be the earliest event indicative of oligodendroglial specification and we propose, based on the expression of plp/dm-20 transcript, a ventricular map of the foci at which oligodendrocytes originate. In addition, we document the precocious segregation, from E5, of plp/dm-20+ and PDGFRα+ oligodendroglial cells in the subventricular and mantle layers of the brain
Functional Analysis of Placental 57-kDa Ca2+-Binding Protein: Overexpression and Downregulation in a Trophoblastic Cell Line

AbstractThe placental trophoblastic epithelium functions to transport nutrients needed by the fetus, including calcium, which is required in the greatest amounts during the last third of pregnancy when the majority of fetal skeletal mineralization occurs. The mechanism of placental calcium transport and the developmental changes in the trophoblast that facilitate this process are currently incompletely understood. We have previously identified a 57-kDa, Ca2+-binding protein (CaBP) functionally implicated in placental calcium transport and trophoblast differentiation. In this study we have directly examined the role of CaBP in these processes by (1) recombinantly overexpressing CaBP in an inducible manner and (2) downregulating CaBP expression using antisense technology, using the rat choriocarcinoma cell line Rcho-1 as a trophoblastic cell model system. Our results show that overexpression of CaBP stimulates both cellular calcium uptake and vectorial calcium transport activities in Rcho-1 cells. Those cells stably expressing CaBP also exhibit higher levels of steady-state intracellular calcium and enhanced calcium-buffering ability. In addition, prolonged overexpression of CaBP in Rcho-1 cultures promotes trophoblast differentiation. Conversely, downregulation of CaBP expression had a negative effect on calcium uptake, calcium transport, and trophoblast differentiation in Rcho-1 cells. These data indicate that CaBP plays a direct role in placental calcium transport, functioning both as an intracellular calcium buffer and as a shuttle. These results also support a more direct role for CaBP in the trophoblast differentiation pathway
casanova Plays an Early and Essential Role in Endoderm Formation in Zebrafish

AbstractThe cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs
Maspin Plays an Important Role in Mammary Gland Development

AbstractMaspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular–alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent
Cell Cycling and Cell Enlargement in Developing Leaves of Arabidopsis

AbstractCell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::β-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level
The Drosophila CPEB Homolog, Orb, Is Required for Oskar Protein Expression in Oocytes

AbstractThe establishment of polarity axes in the Drosophila egg and embryo depends upon the localization and on-site expression of maternal mRNAs. The critical step in the targeting of posterior determinants is the localization of oskar (osk) mRNA to the pole and its on-site translation. Osk protein then recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, nanos. Here we have investigated the role of the Drosophila CPEB homolog, the orb gene, in the osk mRNA localization pathway. We demonstrate that the expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orbmel, little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation inorb mutant ovaries are correlated with a reduction in the length of the osk poly(A) tails. We show that osk mRNA is in immunoprecipitable complexes with Orb protein in ovaries and that the osk 3′ UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of osk mRNA and at that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of “masked” mRNAs
The Cortical Endoplasmic Reticulum (ER) of the Mouse Egg: Localization of ER Clusters in Relation to the Generation of Repetitive Calcium Waves

AbstractThe endoplasmic reticulum (ER) of the mature mouse egg consists of a fine tubular network and pronounced accumulations in the cortex. The ER was visualized both in intact eggs and with in vitro preparations of the cortex using the fluorescent lipophilic dye, DiI. Immunofluorescent labeling of the ER in isolated cortical preparations demonstrated that the ER clusters contain inositol 1,4,5-trisphosphate (IP3) receptors, indicating an important involvement in sperm-induced Ca2+ transients, which are triggered by IP3. We imaged the ER during fertilization and the subsequent Ca2+ transients and found that the clusters remained intact throughout this period. Recovery of fluorescence after photobleaching established that the ER clusters are continuous with the reticular ER network and that these structures remain stable and continuous throughout the time of fertilization-induced Ca2+ transients; continuity also remained during IP3 injection. These results indicate that, in contrast to echinoderm eggs, the ER of mouse eggs does not become disrupted when it releases Ca2+at fertilization. The localization and apparent stability of the cortical ER clusters may be important in generating Ca2+ oscillations, which are characteristic of fertilized mammalian eggs. Imaging of intracellular Ca2+ revealed that Ca2+ transients originate in the hemisphere of the egg that contains abundant ER clusters, thus the mouse contains a stable cortical pacemaker responsible for generating Ca2+ waves
Transcription of the Myogenic Regulatory Gene Mef2 in Cardiac, Somatic, and Visceral Muscle Cell Lineages Is Regulated by a Tinman-Dependent Core Enhancer

AbstractThe MADS-box transcription factor MEF2 is expressed specifically in developing cardiac, somatic, and visceral muscle cell lineages during Drosophila embryogenesis and is required for myoblast differentiation and muscle morphogenesis. To define the mechanisms that regulate Mef2 transcription, we have analyzed the Mef2 upstream region for sequences sufficient to recapitulate the expression pattern of the gene in Drosophila embryos. Here we describe a complex enhancer located 5.8 kb upstream of the Drosophila Mef2 gene that controls transcription in cardial cells of the dorsal vessel, a subset of somatic muscle founder cells, and the visceral muscle cells. The core of this enhancer contains two evolutionarily conserved binding sites for the homeodomain protein Tinman (Tin), expressed in developing cardiac, somatic, and visceral muscle lineages. Both Tin binding sites are required for enhancer activity in all three muscle cell lineages. Whereas the 285-bp enhancer core alone is sufficient for expression in cardiac cells, expression in somatic founder cells and visceral muscle is dependent on the core enhancer plus unique flanking sequences that include an evolutionarily conserved E box. These results reveal an essential role for Tin in activation of Mef2 transcription in multiple myogenic lineages and demonstrate that transcriptional activity of Tin is dependent on combinatorial interactions with other factors unique to different muscle cell types
Mutations in ooc-5 and ooc-3 Disrupt Oocyte Formation and the Reestablishment of Asymmetric PAR Protein Localization in Two-Cell Caenorhabditis elegans Embryos

AbstractThe early development of Caenorhabditis elegans embryos is characterized by a series of asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear–centrosome complex. To identify genes involved in the control of spindle orientation, we have screened maternal-effect lethal mutants for alterations in cleavage pattern. Here we describe mutations in ooc-5 and ooc-3, which were isolated on the basis of a nuclear rotation defect in the P1 cell of two-cell embryos. These mutations are novel in that they affect the asymmetric localization of PAR proteins at the two-cell stage, but not at the one-cell stage. In wild-type two-cell embryos, PAR-3 protein is present around the entire periphery of the AB cell and prevents nuclear rotation in this cell. In contrast, PAR-2 functions to allow nuclear rotation in the P1 cell by restricting PAR-3 localization to the anterior periphery of P1. In ooc-5 and ooc-3 mutant embryos, PAR-3 was mislocalized around the periphery of P1, while PAR-2 was reduced or absent. The germ-line-specific P granules were also mislocalized at the two-cell stage. Mutations in ooc-5 and ooc-3 also result in reduced-size oocytes and embryos. However, par-3 ooc double-mutant embryos can exhibit nuclear rotation, indicating that small size per se does not prevent rotation and that PAR-3 mislocalization contributes to the failure of rotation in ooc mutants. We therefore postulate that wild-type ooc-5 and ooc-3 function in oogenesis and in the reestablishment of asymmetric domains of PAR proteins at the two-cell stage
Different Levels, but Not Different Isoforms, of the Drosophila Transcription Factor DMEF2 Affect Distinct Aspects of Muscle Differentiation

Abstractmef2 genes encode alternatively spliced transcription factor isoforms that function in muscle differentiation in both Drosophila and vertebrates. Drosophila mef2 (Dmef2) has been shown to be required for the differentiation of a variety of distinct muscle types. However, many possible aspects of its function in muscle remain unexplored. There has also been no analysis in vivo of the activity of different MEF2 isoforms in any species. Our investigation centred on the role of different levels of DMEF2 in the Drosophila embryo in regulating diverse events of muscle differentiation and on the functional significance of Dmef2 alternative splicing. We used the GAL4/UAS system to both misexpress and overexpress individual DMEF2 isoforms and to rescue the different aspects of the Dmef2 mutant phenotype. Ectopic ectodermal expression of DMEF2 activated muscle gene expression and inhibited epidermal differentiation. Overexpression of DMEF2 in the mesoderm disrupted differentiation of the somatic and visceral muscle and the heart. The use of different DMEF2 levels in the rescue experiments revealed an activity range compatible with differentiation of the different muscle types: the consequence of too little or too much DMEF2 activity was disrupted differentiation. These rescue experiments also revealed that distinct DMEF2 thresholds are required for different properties within a cell and also for different cells within a muscle type and for different muscle types. Finally, each isoform functioned equivalently in these experiments, including in the stringent test of rescue of the Dmef2 mutant phenotype
Expression of HAND Gene Products May Be Sufficient for the Differentiation of Avian Neural Crest-Derived Cells into Catecholaminergic Neurons in Culture

AbstractMembers of the basic helix-loop-helix family of DNA binding proteins have important roles in the development of subpopulations of neural crest-derived neurons. We have cloned the chicken homologues of dHAND (HAND2) and eHAND (HAND1), basic helix-loop-helix DNA binding proteins whose neuronal expression is restricted to sympathetic and enteric neural crest-derived ganglia. Transcripts encoding dHAND and eHAND are expressed in sympathetic ganglia beginning at Hamburger and Hamilton stage 17–18. Antisense blockade of transcripts encoding HAND genes in neural crest-derived cells in vitro results in a significant reduction in neurogenesis. Differentiation of catecholaminergic neurons is also reduced by 52% if the expression of transcripts encoding dHAND and eHAND is reduced using antisense oligonucleotide blockade. The effect on neurogenesis and phenotypic expression of neural crest-derived neurons is specific; blockade of HAND gene expression has no apparent influence on the differentiation in vitro of neural tube-derived neurons. Use of a replication-competent avian retrovirus to constitutively express HAND genes in neural crest-derived cells in vitro, under nonpermissive growth conditions in medium supplemented with 2% chick embryo extract (CEE), induced precocious catecholaminergic differentiation. Constitutive expression of HAND gene products resulted in a significant increase in catecholaminergic differentiation of cells grown in medium supplemented with 10% CEE, a permissive growth condition for catecholaminergic development. These results suggest that the expression by neural crest cells of dHAND and eHAND may be both sufficient and necessary for catecholaminergic phenotypic expression