pinecone/core-2020-05-10-deduplication huggingface dataset:
Development of “Normal” Dermatomes and Somatotopic Maps by “Abnormal” Populations of Cutaneous Neurons

AbstractDuring development, motor and sensory axons grow to peripheral targets with remarkable precision. Whereas much has been learned about the development of motoneuron connectivity, less is known about the regulation of cutaneous innervation. In adults, dorsal root ganglia (DRG) innervate characteristic skin regions, termed dermatomes, and their axons project somatotopically in the dorsal horn. Here, we have investigated whether cutaneous neurons are selectively matched with specific skin regions, and whether peripheral target skin influences the central connections of cutaneous neurons. To address these questions, we shifted limb buds rostrally in chick embryos prior to axon outgrowth, causing DRGs to innervate novel skin regions, and mapped the resulting dermatomes and central projections. Following limb shifts, cutaneous innervation arose from more rostral and from fewer DRGs than normal, but the overall dermatome pattern was preserved. Thus, DRGs parcel out innervation of skin in a consistent manner, with no indication of matching between skin and DRGs. Similarly, cutaneous nerves established a “normal” somatotopic map in the dorsal horn, but in more rostral segments than usual. Thus, the peripheral target skin may influence the pattern of CNS projections, but does not direct cutaneous axons to specific populations of neurons in the dorsal horn
UNC-5 Function Requires Phosphorylation of Cytoplasmic Tyrosine 482, but Its UNC-40-Independent Functions also Require a Region between the ZU-5 and Death Domains

AbstractMembers of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5
Vascular Abnormalities in Mice Lacking the Endothelial Gap Junction Proteins connexin37 and connexin40

AbstractCells within the vascular wall are coupled by gap junctions, allowing for direct intercellular transfer of low molecular weight molecules. Although gap junctions are believed to be important for vascular development and function, their precise roles are not well understood. Mice lacking either connexin37 (Cx37) or connexin40 (Cx40), the predominant gap junction proteins present in vascular endothelium, are viable and exhibit phenotypes that are largely non-blood vessel related. Since Cx37 and Cx40 are coexpressed in endothelial cells and could overlap functionally, some roles of junctional communication may only be revealed by the elimination of both connexins. In this study, we interbreed Cx37 and Cx40 knockout mice to generate Cx37−/−Cx40−/− animals and show that they display severe vascular abnormalities and die perinatally. Cx37−/−Cx40−/− animals exhibit localized hemorrhages in skin, testis, gastrointestinal tissues, and lungs, with pronounced blood vessel dilatation and congestion occurring in some areas. Vascular anomalies were particularly striking in testis and intestine. In testis, abnormal vascular channels were present, with these channels coalescing into a cavernous, endothelium-lined blood pool resembling a hemangioma. These results provide evidence of a critical role for endothelial gap junction-mediated communication in the development and/or functional maintenance of segments of the mouse vasculature
Importin-α2 Is Critically Required for the Assembly of Ring Canals during Drosophila Oogenesis

AbstractThe interstitial deletion D14 affecting the importin-α2 gene of Drosophila, or imp-α2D14, causes recessive female sterility characterized by a block of nurse cell–oocyte transport during oogenesis. In wild-type egg chambers, the Imp-α2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-α2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-α2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-α2D14 ovaries reveals that the block of nurse cell–oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-α2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-α2D14 egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-α2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly
Properties of a Fetal Multipotent Neural Stem Cell (NEP Cell)

AbstractMultipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFRα), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS
Noggin Is Required for Correct Guidance of Dorsal Root Ganglion Axons

AbstractMembers of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal–ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter
Syntaxin 5 Is Required for Cytokinesis and Spermatid Differentiation in Drosophila

AbstractSyntaxin 5 is a Golgi-localized SNARE protein that has been shown to be required for ER–Golgi traffic in yeast and Golgi reassembly following cell division in mammalian cells. Here, we describe the characterization of the Drosophila ortholog of syntaxin 5, dSyx5, and show that, like its mammalian and yeast counterparts, the protein is localized to the Drosophila Golgi and binds to α-SNAP. Null mutations in dSyx5 are larval lethal and demonstrate impaired transport of a GFP-tagged membrane protein. A hypomorphic allele of dSyx5 caused by insertion of an EP element results in impenetrant lethality, and escaping adult flies are male sterile. The male sterility results both from failure of germ cells to complete cytokinesis and from defects in spermatid elongation and maturation. Ectopic expression of dSyx5 from the EP element can rescue the cytokinesis defect, but high levels of expression are required to restore maturation and fertility. Together, these results show that dSyx5 is required for the proper function of the Golgi apparatus and that an efficiently functioning Golgi apparatus is required for the steps leading to the completion of cytokinesis and formation of mature sperm
The Evolution of Chordate Neural Segmentation

AbstractAmphioxus is the closest relative to vertebrates but lacks key vertebrate characters, like rhombomeres, neural crest cells, and the cartilaginous endoskeleton. This reflects major differences in the developmental patterning of neural and mesodermal structures between basal chordates and vertebrates. Here, we analyse the expression pattern of an amphioxus FoxB ortholog and an amphioxus single-minded ortholog to gain insight into the evolution of vertebrate neural segmentation. AmphiFoxB expression shows cryptic segmentation of the cerebral vesicle and hindbrain, suggesting that neuromeric segmentation of the chordate neural tube arose before the origin of the vertebrates. In the forebrain, AmphiFoxB expression combined with AmphiSim and other amphioxus gene expression patterns shows that the cerebral vesicle is divided into several distinct domains: we propose homology between these domains and the subdivided diencephalon and midbrain of vertebrates. In the Hox-expressing region of the amphioxus neural tube that is homologous to the vertebrate hindbrain, AmphiFoxB shows the presence of repeated blocks of cells along the anterior–posterior axis, each aligned with a somite. This and other data lead us to propose a model for the evolution of vertebrate rhombomeric segmentation, in which rhombomere evolution involved the transfer of mechanisms regulating neural segmentation from vertical induction by underlying segmented mesoderm to horizontal induction by graded retinoic acid signalling. A consequence of this would have been that segmentation of vertebrate head mesoderm would no longer have been required, paving the way for the evolution of the unsegmented head mesoderm seen in living vertebrates
The GLH Proteins, Caenorhabditis elegans P Granule Components, Associate with CSN-5 and KGB-1, Proteins Necessary for Fertility, and with ZYX-1, a Predicted Cytoskeletal Protein

AbstractThe GLH proteins belong to a family of four germline RNA helicases in Caenorhabditis elegans. These putative ATP-dependent enzymes localize to the P granules, which are nonmembranous complexes of protein and RNA exclusively found in the cytoplasm of all C. elegans germ cells and germ cell precursors. To determine what proteins the GLHs bind, C. elegans cDNA libraries were screened by the yeast two-hybrid method, using GLHs as bait. Three interacting proteins, CSN-5, KGB-1, and ZYX-1, were identified and further characterized. GST pull-down assays independently established that these proteins bind GLHs. CSN-5 is closely related to the subunit 5 protein of COP9 signalosomes, conserved multiprotein complexes of plants and animals. RNA interference (RNAi) with csn-5 results in sterile worms with small gonads and no oocytes, a defect essentially identical to that produced by RNAi with a combination of glh-1 and glh-4. KGB-1 is a putative JNK MAP kinase that GLHs bind. A kgb-1 deletion strain has a temperature-sensitive, sterile phenotype characterized by the absence of mature oocytes and the presence of trapped, immature oocytes that have undergone endoreplication. ZYX-1 is a LIM domain protein most like vertebrate Zyxin, a cytoskeletal adaptor protein. In C. elegans, while zyx-1 appears to be a single copy gene, neither RNAi depletion nor a zyx-1 deletion strain results in an obvious phenotype. These three conserved proteins are the first members in each of their families reported to associate with germline helicases. Similar to the loss of GLH-1 and GLH-4, loss of either CSN-5 or KGB-1 causes oogenesis to cease, but does not affect the initial assembly of P granules
Spergen-1 Might Be an Adhesive Molecule Associated with Mitochondria in the Middle Piece of Spermatozoa

AbstractSpergen-1, a recently identified molecule specifically expressed in haploid spermatids in testis, is a small protein of 154 amino acids with a mitochondria-targeting signal at the N terminus. To examine the localization of spergen-1 protein in germ cells, we performed immunocytochemistry with the anti-spergen-1 antibody on frozen sections of rat testis and purified spermatozoa. Immunolabeling for spergen-1 was detected in mitochondria of elongating spermatids and of the middle pieces of matured spermatozoa. Immunoelectron microscopy revealed that spergen-1 was localized to the surface of mitochondria in the middle piece of spermatozoa. To investigate the properties of spergen-1, COS-7 cells were transfected with vectors encoding various spergen-1 mutants. The transfection experiments showed that spergen-1 expressed in the cells tended to agglutinate mitochondria and assemble them into aggregations and that the C-terminal region of spergen-1 as well as the N-terminal mitochondrial targeting signal was requisite for induction of mitochondrial aggregation. These results suggest that spergen-1, a mitochondria-associated molecule in spermatozoa, has a property to induce mitochondrial aggregation at least in cultured cells. We hypothesize that spergen-1 might function as an adhesive molecule to assemble mitochondria into the mitochondrial sheath around the outer dense fibers during spermiogenesis
Patterning through Differential Endoreduplication in Epithelial Organogenesis of the Chordate, Oikopleura dioica

AbstractThe contributions that control of cell proliferation and cell growth make to developmental regulation of organ and body size remain poorly explored, particularly with respect to endocycles in polyploid tissues. The epithelium of the marine chordate Oikopleura dioica is composed of a fixed number of cells grouped in territories according to gene-specific expression and nuclear sizes and shapes. As the animal grows 10-fold during the life cycle, epithelial cells increase in size differentially as a function of their spatial position. We show that this cellular pattern reflected differences in ploidy levels ranging from 34 to 1300 C. The diverse ploidy levels in defined cellular fields resulted both from different timing of entry into endocycles and from cell-specific regulation of endocycle lengths. Throughout the life cycle, differential cell size and ploidy increases were accompanied by field-specific profiles of progressive reductions in G-phase duration. Endocycles were asynchronous among cells of a given epithelial territory, but at the resolution of individual cells, both DNA replication timing and ploidy levels were bilaterally symmetric. The transparent, accessible, oikoplastic epithelium is a model of choice for the study of endoreduplication in the context of pattern formation and growth
Pitx2 Distinguishes Subtypes of Terminally Differentiated Neurons in the Developing Mouse Neuroepithelium

AbstractPitx2, a homeodomain transcription factor, is essential for normal development of pituitary, eyes, heart, and teeth. In the developing mouse brain, Pitx2 (Rieg, Ptx2, Otlx2, Brx1) mRNA is expressed in discrete regions of the diencephalon, mesencephalon, and rhombencephalon. While prior reports have provided an overview of the temporal and regional specificity of Pitx2 mRNA expression in the brain, the precise cell types that express PITX2 are not known. In this study, we analyzed Pitx2 mRNA and PITX2 protein expression in individual cells of the developing e10.5–e14.5 mouse CNS using multiple markers of cellular proliferation and differentiation. We identified Pitx2 expression in nestin-positive neural progenitors and in postmitotic, developing neurons. In the diencephalon, PITX2 is expressed in neurons of the zona limitans intrathalamica and mammillary region and in γ-aminobutyric acid (GABA)-producing neurons of the zona incerta. In the mesencephalon, PITX2-labeled nuclei also appear in differentiated neurons, some of which are GABAergic and destined to occupy superior colliculus. Our results suggest that PITX2 expression in postmitotic neurons may contribute to development of GABAergic and other differentiated neuronal phenotypes
Stripe Provides Cues Synergizing with Branchless to Direct Tracheal Cell Migration

AbstractThe Drosophila tracheal system is an interconnected tubular respiratory network, which is formed by directed stereotypic migration and fusion of branches. Cell migration and specification are determined by combinatorial signaling of several morphogens secreted from the ectoderm. We report the discovery of a group of ectodermal cells, marked by Stripe (Sr) expression, that coordinates tracheal cell migration in the dorsoventral axis. Sr, an EGR family transcription factor, is known to regulate muscle migration. In this study, we show that Sr ectodermal cells also provide signals that are utilized for tracheal migration. These cues are separated in the time course of embryonic development. Initially, tendon-precursor cells are in close proximity to the tracheal cells, and later, when tracheal migration is complete, the muscles displace the trachea and attach to the tendon cells. sr-mutant embryos exhibit defects in migration of all tracheal branches. Although the FGF ligand Branchless (Bnl) is expressed in a subset of tendon-precursor cells independently of Sr, Bnl functions cooperatively with proteins induced by Sr in attraction of tracheal branches
A Floor Plate Enhancer of the Zebrafish netrin1 Gene Requires Cyclops (Nodal) Signalling and the Winged Helix Transcription Factor FoxA2

AbstractThe floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3β, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2—given its importance for midline development also in the mouse—may be one such component
Genetic Mechanism for the Stage- and Tissue-Specific Regulation of Steroid Triggered Programmed Cell Death in Drosophila

AbstractSteroid hormones trigger a wide variety of cell-specific responses during animal development, but the mechanisms by which these systemic signals specify either cell division, differentiation, morphogenesis or death remain uncertain. Here, we analyze the function of the steroid-regulated genes βFTZ-F1, BR-C, E74A, and E93 during salivary gland programmed cell death. While mutations in the βFTZ-F1, BR-C, E74A, and E93 genes prevent destruction of salivary glands, only βFTZ-F1 is required for DNA fragmentation. Analyses of BR-C, E74A, and E93 loss-of-function mutants indicate that these genes regulate stage-specific transcription of the rpr, hid, ark, dronc, and crq cell death genes. Ectopic expression of βFTZ-F1 is sufficient to trigger premature cell death of larval salivary glands and ectopic transcription of the rpr, dronc, and crq cell death genes that normally precedes salivary gland cell death. The E93 gene is necessary for ectopic salivary gland cell destruction, and ectopic rpr, dronc, and crq transcription, that is induced by expression of βFTZ-F1. Together, these observations indicate that βFTZ-F1 regulates the timing of hormone-induced cell responses, while E93 functions to specify programmed cell death
Homeotic Genes Autonomously Specify the Anteroposterior Subdivision of the Drosophila Dorsal Vessel into Aorta and Heart

AbstractThe embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Here, we demonstrate that homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. We provide evidence that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. We also show that Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart