pinecone/core-2020-05-10-deduplication huggingface dataset:
The Zona Pellucida-Initiated Acrosome Reaction: Defect Due to Mutations in the Sperm Glycine Receptor/Cl− Channel

AbstractThe mammalian sperm acrosome reaction (AR) is essential to fertilization, and the egg zona pellucida (ZP) is generally believed to be an in vivo initiator of the fertilizing sperm AR. Previously a neuronal glycine receptor/Cl− channel (GlyR) was detected on the plasma membrane of mammalian sperm and earlier pharmacological studies suggested that this receptor/channel is important to the ZP-initiated AR. Here, sperm from mice with mutations in the neuronal GlyR α or β subunits (spasmodic and spastic) were shown to be deficient in their ability to undergo the AR initiated in vitro by glycine or by solubilized ZP from mouse eggs. However, both spontaneous and calcium ionophore (A23187)-initiated AR were unaffected. The ZP-initiated AR in wild-type sperm was maximal after 2 h of capacitation, but capacitation of sperm from spasmodic mice for up to 3 h did not result in significant ZP-initiated AR. Similar results were observed when sperm from wild-type and spastic mice were compared. Testis from mice with the β subunit mutation contained truncated β subunit mRNAs. Moreover, a monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. Our results are consistent with an essential role for the sperm GlyR in the ZP-initiated AR
Phylogeny of the SOX Family of Developmental Transcription Factors Based on Sequence and Structural Indicators

AbstractMembers of the SOX family of transcription factors are found throughout the animal kingdom, are characterized by the presence of a DNA-binding HMG domain, and are involved in a diverse range of developmental processes. Previous attempts to group SOX genes and deduce their structural, functional, and evolutionary relationships have relied largely on complete or partial HMG box sequence of a limited number of genes. In this study, we have used complete HMG domain sequence, full-length protein structure, and gene organization data to study the pattern of evolution within the family. For the first time, a substantial number of invertebrate SOX sequences have been included in the analysis. We find support for subdivision of the family into groups A–H, as has been suggested in some previous studies, and for the assignment of two new groups, I and J. For vertebrate genes, it appears that relatedness as suggested by HMG domain sequence is congruent with relatedness as indicated by overall structure of the full-length protein and intron–exon structure of the genes. Most of the SOX groups identified in vertebrates were represented by a single SOX sequence in each invertebrate species studied. We have named anonymous sequences and, where appropriate, have suggested systematic names for some previously identified sequences. In addition, we identify an HMG domain signature motif which may be considered representative of the SOX family. Based on our data, we propose a robust phylogeny of SOX genes that reflects their evolutionary history in metazoans
Oscillating Expression of c-Hey2 in the Presomitic Mesoderm Suggests That the Segmentation Clock May Use Combinatorial Signaling through Multiple Interacting bHLH Factors

AbstractVertebrate somitogenesis comprises the generation of a temporal periodicity, the establishment of anteroposterior compartment identity, and the translation of the temporal periodicity into the metameric pattern of somites. Molecular players at each of these steps are beginning to be identified. Especially, members of the Notch signaling cascade appear to be involved in setting up the somitogenesis clock and subsequent events. We had previously demonstrated specific expression of the mHey1 and mHey2 basic helix-loop-helix (bHLH) factors during somitogenesis. Here we show that perturbed Notch signaling in Dll1 and Notch1 knockout mutants affects this expression in the presomitic mesoderm (PSM) and the somites. In the caudal PSM, however, mHey2 expression is maintained and thus is likely to be independent of Notch signaling. Furthermore, we analysed the dynamic expression of the respective chicken c-Hey1 and c-Hey2 genes during somitogenesis. Not only is c-Hey2 rhythmically expressed across the chicken presomitic mesoderm like c-hairy1, but its transcription is similarly independent of de novo protein synthesis. In contrast, the dynamic expression of c-Hey1 is restricted to the anterior segmental plate. Both c-Hey genes are coexpressed with c-hairy1 in the posterior somite half. Further in vitro and in vivo interaction assays demonstrated direct homo- and heterodimerisation between these hairy-related bHLH proteins, suggesting a combinatorial action in both the generation of a temporal periodicity and the anterior–posterior somite compartmentalisation
Stem Cell Factor Functions as a Survival Factor for Mature Leydig Cells and a Growth Factor for Precursor Leydig Cells after Ethylene Dimethane Sulfonate Treatment: Implication of a Role of the Stem Cell Factor/c-Kit System in Leydig Cell Development

AbstractThe significance of the interaction between Sertoli cell-produced stem cell factor (SCF) and its receptor, c-kit, on Leydig cells (LCs) during LC development and differentiation is unknown. In the present study, we investigated the potential role of the SCF/c-kit system in LC apoptosis and precursor LC proliferation after ethylene dimethane sulfonate (EDS) treatment in rats. A function-blocking anti-c-kit antibody, ACK-2, was used to block SCF/c-kit interaction at four time points, corresponding to the peak of LC apoptosis and three waves of proliferation of precursor LCs. Blockade of SCF/c-kit interaction by ACK-2 accelerated LC apoptosis and inhibited proliferation of precursor LCs during the first two waves of precursor LC proliferation around days 3–4 and day 10, but not the third wave of precursor LC proliferation around day 20 after EDS treatment. The data suggest that the soluble SCF might act as a survival factor for mature LCs and a growth factor for precursor LCs after EDS-induced LC depletion. This is also supported by a close correlation between the oscillating levels of soluble SCF mRNA and the profiles of LC apoptosis and regeneration. Since regeneration of the LC population after EDS treatment resembles the development of adult-type LCs during prepubertal life, the present findings imply that soluble SCF might participate in regulation of the formation of the LC population during testicular development. Our data also support a model in which delicate and reciprocal regulation exists between soluble SCF production by Sertoli cells, testosterone production by LCs, and pituitary gonadotropins
Endothelins Control the Timing of Schwann Cell Generation in Vitro and in Vivo

AbstractSchwann cell precursors, derivatives of the neural crest, generate Schwann cells in a process that is tightly timed, well characterized, and directly controlled by axonal signals, in particular β-neuregulins. Here we provide evidence that endothelins (ETs) are also important for survival and lineage progression in this system. We show that ETs promote rat Schwann cell precursor survival in vitro without stimulation of DNA synthesis. Using ET receptor agonists and antagonists, we demonstrate that this action of ET is mediated by the ETB receptor. RT-PCR reveals the presence of ET and ET receptor mRNA in the developing rat PNS. We showed previously that in vitro β-neuregulins promote the generation of Schwann cells from precursors on schedule and that this process can be accelerated by fibroblast growth factor 2. Here we show that although ETs promote long-term precursor survival the transition of precursors to Schwann cells is delayed. Moreover, ETs block the maturation effects of β-neuregulins. In spotting lethal rats, in which functional ETB receptors are absent, we find accelerated expression of the Schwann cell marker S100 in developing nerves. These observations indicate that complex growth factor interactions control the timing of Schwann cell development in embryonic nerves and that ETs act as negative regulators of Schwann cell generation
Ras-Mediated FGF Signaling Is Required for the Formation of Posterior but Not Anterior Neural Tissue in Xenopus laevis

AbstractFibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior–posterior neural patterning, we have employed the dominant negative Ras mutant, N17Ras, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction
The Alternative Migratory Pathways of the Drosophila Tracheal Cells Are Associated with Distinct Subsets of Mesodermal Cells

AbstractThe Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. Here we analyze how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. We find that cells in different branches exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. We also find that alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and propose a model for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints
E2F1 and p53 Are Dispensable, whereas p21Waf1/Cip1 Cooperates with Rb to Restrict Endoreduplication and Apoptosis during Skeletal Myogenesis

AbstractWe describe temporal and genetic analyses of partially rescued Rb mutant fetuses, mgRb:Rb−/−, that survive to birth and reveal specific defects in skeletal muscle differentiation. We show that in the absence of Rb, these fetuses exhibit increased apoptosis, bona fide endoreduplication, and incomplete differentiation throughout terminal myogenesis. These defects were further augmented in composite mutant fetuses, mgRb:Rb−/−:p21−/−, lacking both Rb and the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Although E2F1 and p53 mediate ectopic DNA synthesis and cell death in several tissues in Rb mutant embryos, both endoreduplication and apoptosis persisted in mgRb:Rb−/−:E2F1−/− and mgRb:Rb−/−:p53−/− compound mutant muscles. Thus, combined inactivation of Rb and p21Waf1/Cip1 augments endoreduplication and apoptosis, whereas E2F1 and p53 are dispensable during aberrant myogenesis in Rb-deficient fetuses
Identification of a Novel Isoform of Poly(A) Polymerase, TPAP, Specifically Present in the Cytoplasm of Spermatogenic Cells

AbstractWe have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)12 RNA primer in the presence of MnCl2. Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs
Patterns of MHR3 Expression in the Epidermis during a Larval Molt of the Tobacco Hornworm Manduca sexta

AbstractMHR3, an ecdysone-induced transcription factor, was shown to appear in the abdominal epidermis of the tobacco hornworm Manduca sexta in a pattern-specific manner as the 20-hydroxyecdysone (20E) titer rises for the larval molt. The crochet epidermis that forms the hooked setae on the proleg is first to show MHR3 mRNA and protein followed sequentially by the spiracle, the dorsal intrasegmental annuli, the interannular regions, and finally the trichogen and tormogen cells. The protein appears in the nuclei about 8 h before the onset of cuticle formation, is present during the outgrowth of the setae, and disappears after epicuticle formation. In vitro studies showed that MHR3 mRNA induction in the crochet epidermis by 20E was more sensitive (EC50 = 10−6 M; 50% induction by 2 h exposure to 4 × 10−6 M 20E) and did not require protein synthesis for maximal accumulation compared to the dorsal epidermis. The ecdysone receptor complex is present in both tissues at the outset of the molt and therefore is not a determining factor in these responses. Thus, in addition to the ecdysone receptor complex, region-specific factors govern both sensitivity and timing of responsiveness of MHR3 to 20E to ensure that this transcription factor will be present when needed for its differentiative role
Midline Nasal Tissue Influences Nestin Expression in Nasal-Placode-Derived Luteinizing Hormone-Releasing Hormone Neurons during Development

AbstractNeurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode
The let-268 Locus of Caenorhabditis elegans Encodes a Procollagen Lysyl Hydroxylase That Is Essential for Type IV Collagen Secretion

AbstractBasement membranes are thin sheets of specialized extracellular matrix molecules that are important for supplying mechanical support and for providing an interactive surface for cell morphology. Prior to secretion and assembly, basement membrane molecules undergo intracellular processing, which is essential for their function. We have identified several mutations in a procollagen processing enzyme, lysyl hydroxylase (let-268). The Caenorhabditis elegans lysyl hydroxylase is highly similar to the vertebrate lysyl hydroxylase, containing all essential motifs required for enzymatic activity, and is the only lysyl hydroxylase found in the C. elegans sequenced genome. In the absence of C. elegans lysyl hydroxylase, type IV collagen is expressed; however, it is retained within the type IV collagen-producing cells. This observation indicates that in let-268 mutants the processing and secretion of type IV collagen is disrupted. Our examination of the body wall muscle in these mutant animals reveals normal myofilament assembly prior to contraction. However, once body wall muscle contraction commences the muscle cells separate from the underlying epidermal layer (the hypodermis) and the myofilaments become disorganized. These observations indicate that type IV collagen is required in the basement membrane for mechanical support and not for organogenesis of the body wall muscle
Analysis of Upstream Elements in the HuC Promoter Leads to the Establishment of Transgenic Zebrafish with Fluorescent Neurons

AbstractHuC encodes an RNA binding protein homologous to Drosophila elav that serves as an excellent early marker for differentiating neurons. We have characterized the promoter of the zebrafish HuC gene by examining the ability of 5′-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos. We determined that 2.8 kb of the 5′-flanking sequence is sufficient to restrict GFP gene expression to neurons. The core promoter spans 251 base pairs and contains a CCAAT box and one SP1 sequence but no TATA box is present near the transcription start site. A putative MyT1 binding site and at least 17 E-box sequences are necessary to maintain the neuronal specificity of HuC expression. Interestingly, sequential removal of the putative MyT1 binding site and 14 distal E boxes does not appear to abolish neuronal expression; rather, it leads to a progressive expansion of GFP expression into muscle cells. Further removal of the three proximal E boxes eliminates neuronal and muscle specificity of GFP expression and leads to ubiquitous expression of GFP in the whole body. Identification of key components of the HuC promoter has led to the establishment of a stable zebrafish transgenic line (HuC-GFP) in which GFP is expressed specifically in neurons. We crossed mind bomb (mib) fish with this line to visualize their neurogenic phenotype in live mib−/− mutant embryos. This cross illustrates howHuC-GFP fish could be used in the future to identify and analyze zebrafish mutants with an aberrant pattern of early neurons
Mutational Analysis of Endothelin Receptor b1 (rose) during Neural Crest and Pigment Pattern Development in the Zebrafish Danio rerio

AbstractPigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio
The Zinc Finger Protein Schnurri Acts as a Smad Partner in Mediating the Transcriptional Response to Decapentaplegic

AbstractIn Drosophila, a BMP-related ligand Decapentaplegic (Dpp) is essential for cell fate specification during embryogenesis and in imaginal disc development. Dpp signaling culminates in the phosphorylation and nuclear translocation of Mothers against dpp (Mad), a receptor-specific Smad that can bind DNA and regulate the transcription of Dpp-responsive genes. Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study we show that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore we find that Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes we developed a cell culture assay and show that Shn and Mad act synergistically to induce transcription. Our results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor