A Myelin Proteolipid Protein-LacZ Fusion Protein Is Developmentally Regulated and Targeted to the Myelin Membrane in Transgenic Mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of β-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH_2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane

Phase-Contrast OCT Imaging of Transverse Flows in the Mouse Retina and Choroid

Purpose. To test the hypothesis that a novel phase-contrast optical coherence tomography (OCT) system can image retinal and choroidal vessels in the living mouse. Methods. A high-speed spectral domain optical coherence tomography (SDOCT) system, which measures the reflections for the entire depth of the retina at once with each axial scan (A-scan), was developed for mouse retinal imaging. Acquiring multiple A-scans over a transverse line across the mouse retina offers a two-dimensional cross-sectional image (B-scan); several neighboring B-scans can be assembled into a three-dimensional OCT image. To visualize mobility and transverse flow in retinal vessels, the statistical variance of phase for each location was calculated from multiple B-scans acquired successively for the same retinal cross-section. Such measures of phase variance offer a direct measure of motions over a large dynamic range of flow velocities. Results. Three-dimensional phase-contrast images of the live mouse retina were created using multiple two-dimensional cross-sectional image slices through the retina. For the data presented here, each cross-sectional phase-contrast slice resulted from five images of 100 or 200 transverse pixels, acquired over 25 ms or 50 ms, respectively. The approach offered clear identification of motion regions at different depths, including flow in the retinal microvasculature and in the choroidal vessels. Conclusions. Phase-contrast OCT enables three-dimensional visualization of retinal and choroidal vasculature in vivo

Identification of an embryonic isoform of myelin basic protein that is expressed widely in the mouse embryo

We have identified a myelin basic protein (MBP) isoform in mouse embryos that includes an exon upstream of the usual transcription initiation site. This isoform, embryonic-neonatal MBP (E-MBP), is expressed at the protein level in the embryonic nervous system at a time when other MBP isoforms are not detected. In addition to the central and peripheral nervous systems of the embryo and neonate, the thymus, spleen, and testes also express E-MBP at the protein level. The expression of E-MBP in cell types distinct from the nervous system strongly suggests that this MBP isoform has a role apart from the formation of myelin

Visualizing Diffusion Tensor Images of the Mouse Spinal Cord

Within biological systems water molecules undergo continuous stochastic Brownian motion. The rate of this diffusion can give clues to the structure of underlying tissues. In some tissues the rate is anisotropic - faster in some directions than others. Diffusion-rate images are second-order tensor fields and can be calculated from diffusion-weighted magnetic resonance images. A 2D diffusion tensor image (DTI) and an associated anatomical scalar field, created during the tensor calculation, define seven dependent values at each spatial location. Understanding the interrelationships among these values is necessary to understand the data. We present two new methods for visually representing DTIs. The first method displays an array of ellipsoids where the shape of each ellipsoid represents one tensor value. The novel aspect of this representation is that the ellipsoids are all normalized to approximately the same size so that they can be displayed in context. The second method uses concepts from oil painting to represent the seven-valued data with multiple layers of varying brush strokes. Both methods successfully display most or all of the information in DTIs and provide exploratory methods for understanding them. The ellipsoid method has a simpler interpretation and explanation than the painting-motivated method; the painting-motivated method displays more of the information and is easier to read quantitatively. We demonstrate the methods on images of the mouse spinal cord. The visualizations show significant differences between spinal cords from mice suffering from Experimental Allergic Encephalomyelitis (EAE) and spinal cords from wild-type mice. The differences are consistent with pathology differences shown histologically and suggest that our new non-invasive imaging methodology and visualization of the results could have early diagnostic value for neurodegenerative diseases

Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo

Formation of Compact Myelin Is Required for Maturation of the Axonal Cytoskeleton

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons

Formation of Compact Myelin Is Required for Maturation of the Axonal Cytoskeleton

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons

Statistical diffusion tensor histology reveals regional dysmyelination effects in the shiverer mouse mutant

Shiverer is an important model of central nervous system dysmyelination characterized by a deletion in the gene encoding myelin basic protein with relevance to human dysmyelinating and demyelinating diseases. Perfusion fixed brains from shiverer mutant (C3Fe.SWV Mbp^(shi)/Mbp^(shi)n = 6) and background control (C3HeB.FeJ, n = 6) mice were compared using contrast enhanced volumetric diffusion tensor magnetic resonance microscopy with a nominal isotropic spatial resolution of 80 mum. Images were accurately coregistered using non-linear warping allowing voxel-wise statistical parametric mapping of tensor invariant differences between control and shiverer groups. Highly significant differences in the tensor trace and both the axial and radial diffusivity were observed within the major white matter tracts and in the thalamus, midbrain, brainstem and cerebellar white matter, consistent with a high density of myelinated axons within these regions. The fractional anisotropy was found to be much less sensitive than the trace and eigenvalues to dysmyelination and associated microanatomic changes

Region-Specific Myelin Pathology in Mice Lacking the Golli Products of the Myelin Basic Protein Gene

The myelin basic protein (MBP) gene encodes two families of proteins, the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. In this study, targeted ablation of the golli-MBPs, but not the classic MBPs, resulted in a distinct phenotype unlike that of knock-outs (KOs) of the classic MBPs or other myelin proteins. Although the golli KO animals did not display an overt dysmyelinating phenotype, they did exhibit delayed and/or hypomyelination in selected areas of the brain, such as the visual cortex and the optic nerve, as determined by Northern and Western blots and immunohistochemical analysis with myelin protein markers. Hypomyelination in some areas, such as the visual cortex, persisted into adulthood. Ultrastructural analysis of the KOs confirmed both the delay and hypomyelination and revealed abnormalities in myelin structure and in some oligodendrocytes. Abnormal visual-evoked potentials indicated that the hypomyelination in the visual cortex had functional consequences in the golli KO brain. Evidence that the abnormal myelination in these animals was a consequence of intrinsic problems with the oligodendrocyte was indicated by an impaired ability of oligodendrocytes to form myelin sheets in culture and by the presence of abnormal Ca^(2+) transients in purified cortical oligodendrocytes studied in vitro. The Ca^(2+) results reported in this study complement previous results implicating golli proteins in modulating intracellular signaling in T-cells. Together, all these findings suggest a role for golli proteins in oligodendrocyte differentiation, migration, and/or myelin elaboration in the brain