We have developed a sensitized screen to identify genes involved in gene silencing, using random N-ethyl-N-nitrosourea mutagenesis on mice carrying a variegating GFP transgene. The dominant screen has produced six mutant lines, including both suppressors and enhancers of variegation. All are semidominant and five of the six are homozygous embryonic lethal. In one case, the homozygous lethality depends on sex: homozygous females die at midgestation and display abnormal DNA methylation of the X chromosome, whereas homozygous males are viable. Linkage analysis reveals that the mutations map to unique chromosomal locations. We have studied the effect of five of the mutations on expression of an endogenous allele known to be sensitive to epigenetic state, agouti viable yellow. In all cases, there is an effect on penetrance, and in most cases, parent of origin and sex-specific effects are detected. This screen has identified genes that are involved in epigenetic reprogramming of the genome, and the behavior of the mutant lines suggests a common mechanism between X inactivation and transgene and retrotransposon silencing. Our findings raise the possibility that the presence or absence of the X chromosome in mammals affects the establishment of the epigenetic state at autosomal loci by acting as a sink for proteins involved in gene silencing. The study demonstrates the power of sensitized screens in the mouse not only for the discovery of novel genes involved in a particular process but also for the elucidation of the biology of that process.
There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.
Perivascular spaces play a pivotal role in the exchange between cerebrospinal and interstitial fluids, and in the clearance of waste in the CNS, yet their precise anatomical components are not well described. The aim of this study was to characterise the ultrastructure of perivascular spaces and their role in the transport of fluid, in the spinal cord of healthy rats, using transmission electron microscopy. The distribution of cerebrospinal fluid tracers injected into the subarachnoid space was studied using light, confocal and electron microscopy. Perivascular spaces were found around arterioles and venules, but not capillaries, throughout the spinal cord white and grey matter. They contained fibroblasts and collagen fibres, and were continuous with the extracellular spaces of the surrounding tissue. At 5 min post injection, tracers were seen in the subarachnoid space, the peripheral white matter, the perivascular spaces, basement membranes, extracellular spaces of the surrounding tissue, and surprisingly, in the lumen of blood vessels, suggesting trans-vascular clearance. These findings point out an unrecognised outflow pathway for CNS fluids, with potential implications for volume regulation in health and disease states, but also clinically for the detection of CNS-derived biomarkers in plasma, the immune response and drug pharmacokinetics.
BackgroundCerebrospinal fluid (CSF) is thought to flow into the brain via perivascular spaces around arteries, where it mixes with interstitial fluid. The precise details concerning fluid outflow remain controversial. Although fluid dynamics have been studied in the brain, little is known about spinal cord fluid inflow and outflow. Understanding the normal fluid physiology of the spinal cord may give insight into the pathogenesis of spinal cord oedema and CSF disorders such as syringomyelia. We therefore aimed to determine the fluid outflow pathways in the rat spinal cord.MethodsA fluorescent tracer, Alexa-Fluor®-647 Ovalbumin, was injected into the extracellular space of either the cervicothoracic lateral white matter or the grey matter in twenty-two Sprague–Dawley rats over 250 s. The rats were sacrificed at 20 or 60 min post injection. Spinal cord segments were sectioned and labelled with vascular antibodies for immunohistochemistry.ResultsFluorescent tracer was distributed over two to three spinal levels adjacent to the injection site. In grey matter injections, tracer spread radially into the white matter. In white matter injections, tracer was confined to and redistributed along the longitudinal axonal fibres. Tracer was conducted towards the pial and ependymal surfaces along vascular structures. There was accumulation of tracer around the adventitia of the intramedullary arteries, veins and capillaries, as well as the extramedullary vessels. A distinct layer of tracer was deposited in the internal basement membrane of the tunica media of arteries. In half the grey matter injections, tracer was detected in the central canal.ConclusionsThese results suggest that in the spinal cord interstitial fluid movement is modulated by tissue diffusivity of grey and white matter. The central canal, and the compartments around or within blood vessels appear to be dominant pathways for fluid drainage in these experiments. There may be regional variations in fluid outflow capacity due to vascular and other anatomical differences between the grey and white matter.
Aquaporin-4 (AQP4) is an astroglial water channel protein that plays an important role in the transmembrane movement of water within the central nervous system. AQP4 has been implicated in numerous pathological conditions involving abnormal fluid accumulation, including spinal cord edema following traumatic injury. AQP4 has not been studied in posttraumatic syringomyelia, a condition that cannot be completely explained by current theories of cerebrospinal fluid dynamics. Alterations of AQP4 expression or function may contribute to the fluid imbalance leading to syrinx formation or enlargement. The aim of this study was to examine AQP4 expression levels and distribution in an animal model of posttraumatic syringomyelia. Immunofluorescence and western blotting were used to assess AQP4 and glial fibrillary acidic protein (GFAP) expression in an excitotoxic amino acid/arachnoiditis model of post-traumatic syringomyelia in SpragueDawley rats. At all time-points, GFAP-positive astrocytes were observed in tissue surrounding syrinx cavities, although western blot analysis demonstrated an overall decrease in GFAP expression, except at the latest stage investigated. AQP4 expression was significantly higher at the level of syrinx at three and six weeks following the initial syrinx induction surgery. Significant increases in AQP4 expression also were observed in the upper cervical cord, rostral to the syrinx except in the acute stage of the condition at the three-day time-point. Immunostaining showed that AQP4 was expressed around all syrinx cavities, most notably adjacent to a mature syrinx (six-and 12-week time-point). This suggests a relationship between AQP4 and fluid accumulation in post-traumatic syringomyelia. However, whether this is a causal relationship or occurs in response to an increase in fluid needs to be established.
In this model of posttraumatic syringomyelia, fluid outflow occurred in a diffuse manner into the surrounding extracellular space and toward the central canal and perivascular spaces. Fluid outflow may be an important consideration in the pathogenesis of syringomyelia and the development of new therapies.
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