Anatomy and Neuroscience - Research Publications

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    SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha 1(II) collagen gene
    Lefebvre, V ; Huang, WD ; Harley, VR ; Goodfellow, PN ; deCrombrugghe, B (AMER SOC MICROBIOLOGY, 1997-04)
    The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.
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    Purinergic mechanisms in the control of gastrointestinal motility
    Bornstein, JC (SPRINGER, 2008-09)
    For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.
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    Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia
    McBrien, NA ; Jobling, AI ; Truong, HT ; Cottriall, CL ; Gentle, A (MOLECULAR VISION, 2009-03-02)
    PURPOSE: Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. METHODS: Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. RESULTS: All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. CONCLUSIONS: This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.
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    EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract
    Dottori, M ; Hartley, L ; Galea, M ; Paxinos, G ; Polizzotto, M ; Kilpatrick, T ; Bartlett, PF ; Murphy, M ; Köntgen, F ; Boyd, AW (NATL ACAD SCIENCES, 1998-10-27)
    Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.
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    Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)
    Sangerman, JI ; Boosalis, MS ; Shen, L ; Haigh, S ; Kane, A ; White, GL ; Perrine, SP ; Faller, DV (American Society of Hematology, 2010-11-19)
    Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction.
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    Cell Type-Specific Thalamic Innervation in a Column of Rat Vibrissal Cortex
    Meyer, HS ; Wimmer, VC ; Hemberger, M ; Bruno, RM ; de Kock, CPJ ; Frick, A ; Sakmann, B ; Helmstaedter, M (OXFORD UNIV PRESS INC, 2010-10)
    This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.
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    Number and Laminar Distribution of Neurons in a Thalamocortical Projection Column of Rat Vibrissal Cortex
    Meyer, HS ; Wimmer, VC ; Oberlaender, M ; de Kock, CPJ ; Sakmann, B ; Helmstaedter, M (OXFORD UNIV PRESS INC, 2010-10)
    This is the second article in a series of three studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). Here, we report the number and distribution of NeuN-positive neurons within the C2, D2, and D3 TC projection columns in P27 rat somatosensory barrel cortex based on an exhaustive identification of 89,834 somata in a 1.15 mm(3) volume of cortex. A single column contained 19,109 ± 444 neurons (17,560 ± 399 when normalized to a standard-size projection column). Neuron density differences along the vertical column axis delineated "cytoarchitectonic" layers. The resulting neuron numbers per layer in the average column were 63 ± 10 (L1), 2039 ± 524 (L2), 3735 ± 905 (L3), 4447 ± 439 (L4), 1737 ± 251 (L5A), 2235 ± 99 (L5B), 3786 ± 168 (L6A), and 1066 ± 170 (L6B). These data were then used to derive the layer-specific action potential (AP) output of a projection column. The estimates confirmed previous reports suggesting that the ensembles of spiny L4 and thick-tufted pyramidal neurons emit the major fraction of APs of a column. The number of APs evoked in a column by a sensory stimulus (principal whisker deflection) was estimated as 4441 within 100 ms post-stimulus.
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    Dimensions of a Projection Column and Architecture of VPM and POm Axons in Rat Vibrissal Cortex
    Wimmer, VC ; Bruno, RM ; de Kock, CPJ ; Kuner, T ; Sakmann, B (OXFORD UNIV PRESS INC, 2010-10)
    This is the first article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). S1 receives 2 major types of TC inputs, lemiscal and paralemniscal. Lemiscal axons arise from the ventral posteromedial nucleus (VPM) of the thalamus, whereas paralemniscal fibers originate in the posteromedial nucleus (POm). While these 2 TC projections are largely complementary in L4, overlap in other cortical layers is still a matter of debate. VPM and POm axons were specifically labeled in the same rat by virus-mediated expression of different fluorescent proteins. We show that columnar and septal projection patterns are maintained throughout most of the cortical depth with a lower degree of separation in infragranular layers, where TC axons form bands along rows. Finally, we present anatomical dimensions of "TC projection domains" for a standard column in S1.
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    BMP receptor signaling is required for postnatal maintenance of articular cartilage
    Rountree, RB ; Schoor, M ; Chen, H ; Marks, ME ; Harley, V ; Mishina, Y ; Kingsley, DM ; Lee Niswander, (PUBLIC LIBRARY SCIENCE, 2004-11)
    Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.
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    Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods.
    Mayer, G ; Harzsch, S (Springer Science and Business Media LLC, 2007-07-15)
    BACKGROUND: Onychophora (velvet worms) represent the most basal arthropod group and play a pivotal role in the current discussion on the evolution of nervous systems and segmentation in arthropods. Although there is a wealth of information on the immunolocalization of serotonin (5-hydroxytryptamine, 5-HT) in various euarthropods, as yet no comparable localization data are available for Onychophora. In order to understand how the onychophoran nervous system compares to that of other arthropods, we studied the distribution of serotonin-like immunoreactive neurons and histological characteristics of ventral nerve cords in Metaperipatus blainvillei (Onychophora, Peripatopsidae) and Epiperipatus biolleyi (Onychophora, Peripatidae). RESULTS: We demonstrate that paired leg nerves are the only segmental structures associated with the onychophoran nerve cord. Although the median commissures and peripheral nerves show a repeated pattern, their arrangement is independent from body segments characterized by the position of legs and associated structures. Moreover, the somata of serotonin-like immunoreactive neurons do not show any ordered arrangement in both species studied but are instead scattered throughout the entire length of each nerve cord. We observed neither a serially iterated nor a bilaterally symmetric pattern, which is in contrast to the strictly segmental arrangement of serotonergic neurons in other arthropods. CONCLUSION: Our histological findings and immunolocalization experiments highlight the medullary organization of the onychophoran nerve cord and argue against segmental ganglia of the typical euarthropodan type being an ancestral feature of Onychophora. These results contradict a priori assumptions of segmental ganglia being an ancestral feature of arthropods and, thus, weaken the traditional Articulata hypothesis, which proposes a sistergroup relationship of Annelida and Arthropoda.