Phenotypic and genetic analysis of the hindshaker mutation A thesis presented to the Faculty of Veterinary Medicine, University of Glasgow for the degree of Doctor of Philosophy December 1997 © Helen Anne King ProQuest Number: 11007714 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11007714 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 G & 2Q 7J UNIVERSITY VMM no 17 fcoK GLASGOW' u n iv e r sit y l ib r a r y ABSTRACT Hindshaker {hsh) is a novel spontaneous murine mutation with autosomal recessive inheritance. Homozygous animals of both sexes exhibited a tremor of the hind end which commenced at about 2 weeks of age and largely disappeared in animals older than 6 weeks. The severity of tremor varied between mice of the same age and a small proportion did not develop a phenotype at all. Seizures, infertility and premature death were not features of the mutation. There was hypomyelination affecting predominantly the spinal cord, although optic nerves and brain were involved to a much lesser degree. The defect of thinly myelinated and naked axons was maximal at 20 days and largely resolved with time so that in the adult, most axons were myelinated. Myelin appeared structurally normal and immunocytochemistry indicated the presence of a panel of myelin proteins. Although distribution of oligodendrocytes in the spinal cord was similar to normal, hypomyelination appeared to be due, at least in part, to a paucity of mature oligodendrocytes that was most profound in the spinal cord. The partial resolution of the myelin deficit was concomitant with an increase in the number of these cells. No ultrastructural abnormalities were found in hsh oligodendrocytes and in vitro studies failed to reveal any differences between the differentiation of 0-2A progenitors obtained from hsh and control mice. No marked astrocytosis was apparent although a microgliosis was detected in older animals. Taken together, the evidence suggests that the hsh mutation does not involve a major structural protein but more probably perturbs an aspect of oligodendrocyte development. The first stage of genetic mapping was undertaken using microsatellite markers and an intersubspecific (CAST/Ei) and two interstrain backcrosses (C57BL/6J and B ALB/cJ). Incomplete penetrance and variation in the expression of the phenotype occurred on these genetic backgrounds due to the action of specific alleles at other non-disease loci. One such modifying locus was identified on proximal chromosome 17. The hsh gene was mapped to a 2cM interval in the middle of chromosome 3; three flanking and five intervening microsatellite markers were identified on a backcross population of 184 mice. There were no obvious candidate genes found on examination of composite genetic maps although Cnp2 apparently lies 2cM proximal to the interval of interest and remains an interesting contender. Further fine mapping now awaits the generation of vital recombination breakpoints to delimit the hsh gene to a sufficiently small interval for positional cloning by conti g construction. To George and Mum List of contents Abstract ................................................................................................................................ i Dedication ...........................................................................................................................ii List of contents .................................................................................................................iii List of figures ................................................................................................................... xi List of tables.....................................................................................................................xv Declaration ..................................................................................................................... xvi Acknowledgements .......................................................................................................xvii 1. Introduction................................................................................................................1 1.1 General introduction ............................................................................................... 2 1.2 Development in the central nervous system ........................................... 2 1.3 Cells of the CNS ......................................................................................................5 1.3.1 Oligodendrocytes ............................................................................................. 5 1.3.2 Astrocytes ....................................................................................................... 17 1.3.3 Microglia ........................................................................................................ 18 1.4 Formation and morphology of the myelin sheath ...............................................19 1.5 Myelinogenesis ......................................................................................................21 1.6 Molecular biology of the myelin sheath ............................................................. 22 1.6.1 Lipids .............................................................................................................. 22 1.6.2 Proteins ........................................................................................................... 22 1.7 Review of CNS myelin mutants .......................................................................... 27 1.7.1 Introduction ....................................................................................................27 1.7.2 shiverer and myelin-deficient ....................................................................... 27 1.7.3 quaking ...........................................................................................................28 1.7.4 Pip mutants .................................................................................................... 29 1.8 Genetic mapping in the mouse ............................................................................ 34 1.8.1 Introduction ................................................................................................... 34 1.8.2 Mouse taxonomy ...........................................................................................34 1.8.3 The mouse genome and mutant loci ............................................................ 37 1.8.4 Linkage mapping in the mouse .....................................................................37 1.8.5 Modifiers of spontaneous mutants ............................................................... 42 1.9 Aims of thesis ........................................................................................................43 2. Materials and Methods ..........................................................................................44 2.1 Tissue fixation .......................................................................................................45 2.1.1 Fixatives.........................................................................................................45 2.1.2 Fixation techniques ........................................................................................45 2.2 Tissue processing and cutting .............................................................................. 46 2.2.1 Paraffin processing and sectioning .............................................................. 46 2.2.2 Resin processing and sectioning...................................................................46 2.2.3 Cryopreservation and sectioning ................................................................. 47 2.3 Staining techniques ............................................................................................... 47 2.3.1 Light microscopy ...........................................................................................47 2.3.2 Electron microscopy ......................................................................................47 2.4 Morphological studies .............................................. 48 2.4.1 Quantification of glia .................................................................................... 48 2.4.2 Dead cell density ...........................................................................................48 iv 2.4.3 Myelin volume ................................................................................................48 2.4.4 Morphometry ..................................................................................................49 2.4.5 Classification of fibre type ........................................................................... 49 2.4.6 Statistical analysis ......................................................................................... 49 2.5 Immunocytochemistry .......................................................................................... 50 2.5.1 Peroxidase-anti-peroxidase