Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

Glutamic Acid Decarboxylase (GAD) is a rate-limiting enzyme responsible for synthesis of the inhibitory neurotransmitter GABA. Dopaminergic denervation in rodents by unilateral injections of 6-OHDA or MPTP causes an increase in Gad67 mRNA in the striatum, which is further exacerbated by administration of L-Dopa (Horvath et al., 2011; Katz et al., 2005 Bacci et al., 2002). Denervation of nigrostriatal neurons is the key pathological hallmark of Parkinson's disease, which results in hypokinetic movement and rigidity. Medium spiny projection neurons of the striatum comprise 95% of the neuronal population and utilize Gad67 (encoded by the Gad1 gene) for the synthesis of basal levels of GABA. The contribution of Gad67 to GABA signaling in medium spiny projection neurons in the striatum has not been thoroughly understood in normal or Parkinsonian states. Mice with a deletion in Gad67 in Gpr88 expressing neurons were generated by crossing mice with a floxed exon 2 of Gad1 with mice expressing Cre recombinase under the control of the Gpr88 promoter. The aim of this study was first, to characterize mice with a deletion in striatal Gad67 by immunohistochmical, electriophysiological and behavioral examination to determine whether Gad67 expression contributes to sensorimotor and learning tasks. And next, to investigate whether a downregulation in striatal Gad67 would decrease dyskinesia and affect the impaired motor symptoms following dopaminergic denervation with a unilateral 6-OHDA lesion and subsequent treatment with L-Dopa. In this study, neuronal Gpr88 expression was indicated by GFP reporter expression, which resulted from Cre-mediated excision of exon 2 of the Gad1 gene. Gpr88 expression was confirmed in the striatum, olfactory tubercle, cortex and brain stem. Furthermore, Gpr88 was confined to striatonigral and striatopallidal MSNs in the striatum. Additionally, Cre-mediated GFP reporter expression indicated that Gpr88 expression occurs throughout various brain regions, including the motor and visual areas of the cortex, amygdala, hippocampus and cerebellum during development. The developmental expression of Gpr88 seems to be a highly regulated process that occurs throughout the brain. In the conditional knockout mouse, deleting striatal Gad67 resulted in an upregualtion of Gad67 in the globus pallidus and downregulation in the substantia nigra. The changes in Gad67 expression indicate the effects of inactivating GABAergic signaling in striatonigral and striatopallidal MSNs in the direct and indirect pathways. Mice with a deletion in striatal Gad67 demonstrated compromised performance in spatial learning in the Morris water maze, suggesting that GABAergic striatal signaling in the direct and indirect pathways accounts for cue-based learning and spatial memory. However, inactivation of GABAergic signaling in striatonigral and striatopallidal MSNs does not account for motor deficits such as bradykinesia, akinesia or hypokinesia in intact mice; instead it perpetuates hyperkinetic motor activity. In the second experiment of this study, dopaminergic denervation by a unilateral 6-OHDA lesion induced bradykinesia and hypokinetic motor behavior, as demonstrated by impaired performance in the rota-rod and pole test. Additionally, L-Dopa administration to 6-OHDA lesioned mice evoked abnormal involuntary movements (AIMs) to the same degree in all dyskinetic mice. A deletion in striatal Gad67 did not decrease symptoms of dyskinesia, nor cause a lessening of motor impairment caused by dopaminergic denervation. Complete inactivation of the indirect pathway is believed to limit the inhibition of unwanted actions and may perpetuate dyskinesia, even when striatonigral MSNs of the direct pathway are inactive.

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24669号 / 農博第2552号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5450(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 菅瀬 謙治, 教授 小川 順, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

L’ostéopétrose est une pathologie osseuse caractérisée par des os denses et fragiles principalement due à l’incapacité des ostéoclastes, cellules d’origine hématopoïétique, à résorber le tissu osseux. La forme la plus sévère de cette maladie génétique est l’ostéopétrose autosomale récessive infantile due à une mutation du gène Ostm1 (Protéine transmembranaire de type 1 associée à l'ostéopétrose). Le gène Ostm1 est exprimé principalement dans la lignée des cellules hématopoïétiques, mais aussi dans le système nerveux central et les mélanocytes. Cette mutation développe plusieurs symptômes comme l’apparition d’une couleur de pelage gris chez la souris, une anémie sévère, une sensibilité aux infections et des troubles neuronaux chez l’homme et la souris. Afin de mieux comprendre cette maladie, nous avons généré des souris transgéniques sur un fond génétique grey-lethal (gl) dans lesquelles l’expression d’Ostm1 est ciblée à un tissu spécifique. Nous avons caractérisé le gène Ostm1 responsable de la mutation ostéopétrotique spontanée gl chez la souris. La complémentation fonctionnelle des défauts hématopoïétiques a été obtenue dans les souris transgéniques PU.1-Ostm1-gl/gl mais ces souris meurent prématurément avec une neurodegénérescence sévère. Cette perte cellulaire affecte le système nerveux central dans son ensemble incluant la rétine. Ce mémoire porte sur le but d’établir le profil d’expression du gène Ostm1 dans la rétine puisque la perte du gène entraine une dégénérescence rétinienne. Pour définir le rôle d’Ostm1 dans la rétine, nous avons caractérisé son expression dans ce tissu (organe). Des analyses PCR, démontrent une expression d’Ostm1 dans l’œil total et enrichie dans la neurorétine et dans l’épithélium pigmentée (RPE). Après avoir caractérisé avec des marqueurs protéiques spécifiques les sous populations cellulaires de la rétine, in situ hybridation détecte l’expression préférentielle d’Ostm1 dans l’épithélium pigmentée (RPE) et la couche nucléaire interne (INL). Basé sur ce profil d’expression, nous avons induit dans un premier temps la perte de fonction d’Ostm1 spécifiquement dans le RPE. Dans un premier temps nous avons vérifié que l’expression de la recombinasse Cre seule n’est pas toxique. Nous avons ensuite induit la perte d’expression d’Ostm1 dans ces cellules et démontré que la perte d’Ostm1 dans le RPE se traduit par une perte graduelle des photorécepteurs avec l’âge. Ces résultats préliminaires suggèrent que l’expression post-natale d’Ostm1 dans le RPE est essentielle au maintien de l’homéostasie des photorécepteurs dans la rétine. / Osteopetrosis is a disease characterized by high bone density and fragility principally caused by impaired activity of osteoclasts, which are cells that reside in bone and dissolve bone tissue. The most severe form of osteopetrosis is infantile autosomal recessive osteopetrosis (ARO) which is caused by mutations in genes Ostm1. As Ostm1(osteopetrosis-associated transmembrane protein 1) is expressed in multiple hematopoietic stem cell lineages, melanocytes and the nervous system, mutations in Ostm1 can cause coat color change in mice as well as bone fragility, anemia, infections and neuronal disorders in humans and mice. To further the understanding of these conditions linked with Ostm1 loss, multiple tissue specific Ostm1 transgenic mice over an Ostm1 knockout (gl/gl) background were constructed. To better understand this disease, we characterized the Ostm1 gene responsible for the spontaneous osteopetrotic mutation grey- lethal (gl) in mice. Functional complementation of hematopoietic defects was obtained in PU.1-Ostm1-gl/gl transgenic mice, but these mice die prematurely with severe neurodegeneration. This indicates that Ostm1 has a crucial role in neuronal and retinal health. As a result, we wished to establish an expression profile of Ostm1 in all the layers of the retina to further decipher the role of Ostm1 in the retina. Polymerase chain reaction (PCR) of reverse-transcribed mRNA of separated sections of the eye demonstrate that Ostm1 is expressed in the whole eye, neuroretina and retinal pigmented epithelium (RPE). Further specific expression analyses were performed by in- situ hybridization which showed that Ostm1 is expressed specifically in the inner nuclear layer of the neuroretina as well as in the RPE. Based on this tissue expression pattern, we have constructed, for the first time, an RPE specific knockdown of Ostm1 expression and verified that the expression of Cre recombinase in this tissue is not toxic. The reduction of Ostm1 in the RPE of the eye resulted in gradual loss of photoreceptors of the retina. These preliminary results suggest that the post-natal expression of Ostm1 in the RPE is essential for maintaining the homeostasis of the photoreceptors of the retina.

Superviseur : Dr. Piet Van Vliet Collaborateurs: Dr. Alexandre Dubrac, Dr. Martin Smith / Résumé INTRODUCTION : Les maladies valvulaires du cœur surviennent dans 2% de la population, impliquant souvent un reflux sanguin dû au rétrécissement de la valve. Nous avons récemment identifié deux familles non apparentées étant atteintes par une sténose aortique. Le séquençage exomique des familles a révélé une mutation entrainant une perte de fonction homozygote pour le gène ADAMTS19. La relation entre la perturbation du gène ADAMTS19 et la sténose a été reproduite et donc confirmée grâce à une souris ADAMTS19-LACZ KO/KO. Cette souris montre également que ADAMTS19 est spécifiquement exprimé dans les cellules valvulaires interstitielles (VICs) dans les valves. Le rôle d’ADAMTS19 durant le développement des valves reste inconnu. Pour analyser le patron d’expression d’ADAMTS19 pendant le développement du cœur, nous avons obtenu un modèle de souris transgénique contenant une CRE-tamoxifen inductible (Cre-ERT2) qui est exprimé sous l’influence du promoteur humain ADAMTS19. HYPOTHESE/OBJECTIF : Nous émettons l’hypothèse que le promoteur humain d’ADAMTS19 inséré dans la souris ADAMTS19-Cre-ERT2 contient toutes les séquences régulatrices permettant d’exprimer le gène ADAMTS19 et que ADAMTS19 est principalement exprimé au niveau des cellules valvulaires interstitielles dans les valves. L’objectif est de caractériser le patron d’expression d’ADAMTS19 en analysant sa distribution durant le développement grâce à une souris reportrice tdTomato. Comme ADAMTS19 est spécifiquement exprimé dans les VICs, cet outil transgénique permettrait d’étudier ces cellules durant le développement. METHODE/RESULTAT : Suite à une étude in silico le promoteur ADAMTS19 est apparu comme extrêmement conservé. Par conséquent, pour analyser l’expression d’ADAMTS19 nous avons obtenu une souris BAC ADAMTS19-Cre-ERT2 contenant la séquence conservée que nous avons croisé avec une souris reportrice tdTomato. Le Tamoxifen est administré aux femelles gestantes par gavage aux jours embryonnaires E9,5, E11,5 ainsi que E13,5, et les cœurs sont extrait a E16,5. Des coupes de cœurs embryonnaires vont permettre d’identifier la localisation et la morphologie des cellules marquées. L’expression d’ADMATS19 dans les cellules valvulaires interstitielles est consistant avec le fait qu’ADAMTS19 est connu pour affecter les valves durant le développent et dans le cas de maladie valvulaire. Cependant, le patron des valves n’est pas reproductible au travers des générations. De plus, nous observons qu’ADAMTS19 est marqué dans des cellules des oreillettes et ventricule dans une lignée et dans une sous population de cellules de l’artère pulmonaire dans une autre. CONCLUSION : L’analyse des séquences de chaque lignée par séquençage permettre d’investiguer la raison de ses différents patrons et de mettre en évidence des régulateurs spécifiques. / BACKGROUND: Valvular heart disease (VHD) occurs in ~2% of the general population, often resulting in reduced or disturbed blood flow. We recently identified two unrelated families with recessive inheritance patterns of progressive polyvalvular heart disease in absence of any clear syndromic phenotype. Exome sequencing revealed homozygous, rare, loss of function (LOF) alleles in both families for the gene ADAMTS19. The relation between ADAMTS19 mutation and aortic stenosis were confirm via an ADAMTS19-LacZ KO/KO mouse model. This model also shows that ADAMTS19 is specific of VICs during valve development. The ADAMTS protein family includes 19 proteases that are involved in matrix remodeling, and tissue homeostasis in development and disease. However, the role of ADAMTS19 specifically during valve development remains unknown. We aim to characterize ADAMTS19 expression using a BAC transgenic ADMTS19-CRERT2 mouse. HYPOTHESE/OBJECTIVE We hypothesize that the BAC used to make the ADMTS19-CRERT2 mouse contains all the regulatory elements to express it and also that its expression will be specific to the VICs. The objective is to establish the ADAMTS19- CREERT2 and therefore to create a new tool to study VICs in vivo. METHODS/RESULTS: In silico analysis of the human and mouse ADAMTS19 genomic regions showed a high level of conservation. Thus, to analyze ADAMTS19 expression patterns during mouse development, we obtained a BAC transgenic mouse model containing a tamoxifen inducible Cre (CreERT2) that is expressed under the influence of the human ADAMTS19 promoter and surrounding genomic region. We crossed males from several lines created in parallel with Rosa-tdTomato reporter females to generate offspring in which expression of the fluorescent tdTomato reporter is activated in ADAMTS19-expressing cells upon tamoxifen administration. Surprisingly, whole mount imaging of embryos induced at E13.5 and isolated at E16.5 revealed strong, but distinct labelling patterns in offspring from different ADAMTS19CreERT2 sublines. Whereas one line exclusively labelled VICs, consistent with ADAMTS19 in situ RNA expression data from the Eurexpress database, another line specifically labelled cells in atrial and ventricular, but not VICs. A third line seems to label only a subset of cells in the pulmonary artery. Labeling of ADAMTS19-positive VICs is consistent with ADAMTS19 affecting valve development and VHD. In addition, we observed exclusive ADAMTS19-dependent labelling in atrial and ventricular cells or in a subset of pulmonary artery cells in two different sublines. CONCLUSION: The distinct expression patterns in offspring from different ADAMTS19-Cre-ERT2 lines indicates that although regulation of ADAMTS19 is conserved between human and mouse, expression in VICs versus other cells may be dependent on mutually exclusive regulatory mechanisms.

The aim of the current study was divided into two separate goals, (i) optimization of a number of PCR-based protocols employed for genotyping transgenic mouse lines and (ii) evaluating a protocol for co-detection of mRNA and its correlated protein in the mouse midbrain. The optimization was performed on PCR protocols for genotyping the following transgenic mouse lines; Dat-Cre, Vglut2-Lox, Vglut2-Cre and Vmat2-Lox. Also, two different polymerases were evaluated parallel to each other – KAPA and Maxima Hot Start. One of the main findings from the PCR optimizations were that for the Vglut2-Lox protocol. By decreasing the annealing temp and increasing the MgCl2 the bands appeared brighter.  For the second part of the project, in-situ hybridization (ISH) was used to detect the mRNA expression with a `non-radioactive in situ hybridization´ protocol, using digoxigenin or fluorescein labelled riboprobes (mRNA probes). To detect the correlated protein a basic immunohistochemistry (IHC) protocol with the use of primary and secondary antibodies was implemented. The combined protocol was tested with Nd6 and Grp markers. Before testing to combined the protocols the ISH protocol was performed alone with riboprobes for Girk2, Lpl and Fst. The combined protocol detected mRNA and protein for both the control marker Th and the Nd6 marker. In conclusions, the optimized PCR protocols were optimal when used with the Maxima Hot Start polymerase and the new combined ISH and IHC protocol worked for markers Th and Nd6.

SLMAP is a tail anchored membrane protein that alternatively splices to generate three isoforms, SLMAP1, SLMAP2 and SLMAP3. Previous studies in our lab have shown that the postnatal cardiac-specific overexpression of SLMAP1 results in intracellular vesicle expansion and enhanced endosomal recycling. I generated a postnatal cardiac-specific knockout model using the Cre-Lox system to nullify all three SLMAP isoforms and further evaluate its role in the mouse myocardium. SLMAP knockdown and knockout mouse hearts were analyzed with western blotting and qPCR. I found that only SLMAP3 was nullified and phenotypic evaluation through echocardiography indicated that young and old SLMAP3 knockout animals showed no remarkable changes in cardiac function. Furthermore, challenge with stressor isoproterenol had a similar response to wildtype and knockout mice in cardiac structure and function. Surprisingly the level of expression of SLMAP1 and SLMAP2 was maintained in the myocardium from SLMAP3 deficient mice. Interestingly the machinery involved in endosomal recycling was not impacted by the loss of SLMAP3. These data indicate that loss of SLMAP3 does not alter cardiac structure and function in the postnatal myocardium in the presence of SLMAP1 and SLMAP2.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24505号 / 医博第4947号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 林 康紀, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM