8 research outputs found
Opposing roles for Hoxa2 and Hoxb2 in hindbrain oligodendrocyte patterning
Oligodendrocytes are the myelin-forming cells of the vertebrate CNS. Little is known about the molecular control of region-specific oligodendrocyte development. Here, we show that oligodendrogenesis in the mouse rostral hindbrain, which is organized in a metameric series of rhombomere-derived (rd) territories, follows a rhombomere-specific pattern, with extensive production of oligodendrocytes in the pontine territory (r4d) and delayed and reduced oligodendrocyte production in the prepontine region (r2d, r3d). We demonstrate that segmental organization of oligodendrocytes is controlled by Hoxgenes, namely Hoxa2 and Hoxb2. Specifically, Hoxa2 loss of function induced a dorsoventral enlargement of the Olig2/Nkx2.2-expressing oligodendrocyte progenitor domain, whereas conditional Hoxa2 overexpression in the Olig2(+) domain inhibited oligodendrogenesis throughout the brain. In contrast, Hoxb2 deletion resulted in a reduction of the pontine oligodendrogenic domain. Compound Hoxa2(-/-)/Hoxb2(-/-) mutant mice displayed the phenotype of Hoxb2(-/-) mutants in territories coexpressing Hoxa2 and Hoxb2 (rd3, rd4), indicating that Hoxb2 antagonizes Hoxa2 during rostral hindbrain oligodendrogenesis. This study provides the first in vivo evidence that Hox genes determine oligodendrocyte regional identity in the mammalian brain
Live imaging of targeted cell ablation in Xenopus:a new model to study demyelination and repair
Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the central nervous system (CNS). In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair
Contribution of demyelination and remyelination in Xenopus laevis
La lignĂ©e transgĂ©nique pMBP-eGFP-NTR, que nous avons gĂ©nĂ©rĂ©e chez Xenopus laevis, permet une ablation conditionnelle des oligodendrocytes myĂ©linisants, dont la consĂ©quence est une dĂ©myĂ©linisation. Dans cette lignĂ©e transgĂ©nique, le transgĂšne est formĂ© par une protĂ©ine de fusion entre le rapporteur GFP (Green Fluorescent Protein= protĂ©ine fluoresçant en vert) et une enzyme de sĂ©lection, la nitrorĂ©ductase dâE. Coli. Cette enzyme, a la propriĂ©tĂ© de rĂ©duire le radical nitrite (NO2) de certains substrats (comme le mĂ©tronidazole) en dĂ©rivĂ© hydroxylamine extrĂȘmement toxique pour la cellule qui lâexprime. Lâexpression de ce transgĂšne est contrĂŽlĂ©e par la portion proximale du gĂšne MBP (myelin basic protein), sĂ©quence rĂ©gulatrice, dont lâĂ©quipe avait dĂ©montrĂ©, chez la souris, quâelle ne sâexprime que dans les oligodendrocytes myĂ©linisants, ce qui câest vĂ©rifiĂ© chez le xĂ©nope. Mon projet se proposait dâĂ©tudier les consĂ©quences de la dĂ©myĂ©linisation et de la remyĂ©linisation dans cette lignĂ©e transgĂ©nique de Xenopus laevis. Mon objectif avait pour but de rĂ©pondre Ă deux questions; Tout dâabord, quâelle est la nature des cellules qui remplacent les oligodendrocytes Ă©liminĂ©s: Nous montrons que les cellules responsables de la remyĂ©linisation sont les prĂ©curseurs des oligodendrocytes (OPCs), cellules GFP nĂ©gatives caractĂ©risĂ©es par lâexpression du facteur de transcription Sox10. Ces cellules OPCs sont dĂ©jĂ prĂ©sentent dans le nerf optique avant lâĂ©vĂ©nement de dĂ©myĂ©linisation. La seconde question visait Ă examiner les consĂ©quences dâune dĂ©myĂ©linisation sur lâarborisation des axones des cellules ganglionnaires de la rĂ©tine. A cette fin nous avons mis au point un outil expĂ©rimental permettant de visualiser lâarborisation des projections tectales des axones des cellules ganglionnaires de la rĂ©tine par microscopie in vivo rĂ©alisĂ©e sur le tĂȘtard au stade 55. Nous montrons que bien que cette arborisation soit plus sensible Ă lâimagerie aprĂšs dĂ©myĂ©linisation, chez le transgĂ©nique que chez le contrĂŽle, cela nâentraine pas de changement de la motilitĂ© de lâarborisation.We have generated a Xenopus laevis transgenic line, pMBP-eGFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of myelin basic protein (MBP) regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by GFP reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. My PhD project is to study the effect of demyelination and remyelination in Xenopus Laevis in this transgenic line. We wish to answer two questions using this transgenic. First, the origin of remyelinating cells that replace the ablated oligodendrocytes. We have shown that, Sox10+ OPCs, which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination. The second question is to examine the effect of demyelination of retinal ganglion cell (RGC) axonal arbor morphology. We developed an experimental set up to image the RGC arbor morphology in an awake stage 55 tadpole in real time. We show that the arbor is more sensitive than the control to imaging but there is no change in motility of the arbor
Contribution of demyelination and remyelination in Xenopus laevis
La lignĂ©e transgĂ©nique pMBP-eGFP-NTR, que nous avons gĂ©nĂ©rĂ©e chez Xenopus laevis, permet une ablation conditionnelle des oligodendrocytes myĂ©linisants, dont la consĂ©quence est une dĂ©myĂ©linisation. Dans cette lignĂ©e transgĂ©nique, le transgĂšne est formĂ© par une protĂ©ine de fusion entre le rapporteur GFP (Green Fluorescent Protein= protĂ©ine fluoresçant en vert) et une enzyme de sĂ©lection, la nitrorĂ©ductase dâE. Coli. Cette enzyme, a la propriĂ©tĂ© de rĂ©duire le radical nitrite (NO2) de certains substrats (comme le mĂ©tronidazole) en dĂ©rivĂ© hydroxylamine extrĂȘmement toxique pour la cellule qui lâexprime. Lâexpression de ce transgĂšne est contrĂŽlĂ©e par la portion proximale du gĂšne MBP (myelin basic protein), sĂ©quence rĂ©gulatrice, dont lâĂ©quipe avait dĂ©montrĂ©, chez la souris, quâelle ne sâexprime que dans les oligodendrocytes myĂ©linisants, ce qui câest vĂ©rifiĂ© chez le xĂ©nope. Mon projet se proposait dâĂ©tudier les consĂ©quences de la dĂ©myĂ©linisation et de la remyĂ©linisation dans cette lignĂ©e transgĂ©nique de Xenopus laevis. Mon objectif avait pour but de rĂ©pondre Ă deux questions; Tout dâabord, quâelle est la nature des cellules qui remplacent les oligodendrocytes Ă©liminĂ©s: Nous montrons que les cellules responsables de la remyĂ©linisation sont les prĂ©curseurs des oligodendrocytes (OPCs), cellules GFP nĂ©gatives caractĂ©risĂ©es par lâexpression du facteur de transcription Sox10. Ces cellules OPCs sont dĂ©jĂ prĂ©sentent dans le nerf optique avant lâĂ©vĂ©nement de dĂ©myĂ©linisation. La seconde question visait Ă examiner les consĂ©quences dâune dĂ©myĂ©linisation sur lâarborisation des axones des cellules ganglionnaires de la rĂ©tine. A cette fin nous avons mis au point un outil expĂ©rimental permettant de visualiser lâarborisation des projections tectales des axones des cellules ganglionnaires de la rĂ©tine par microscopie in vivo rĂ©alisĂ©e sur le tĂȘtard au stade 55. Nous montrons que bien que cette arborisation soit plus sensible Ă lâimagerie aprĂšs dĂ©myĂ©linisation, chez le transgĂ©nique que chez le contrĂŽle, cela nâentraine pas de changement de la motilitĂ© de lâarborisation.We have generated a Xenopus laevis transgenic line, pMBP-eGFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of myelin basic protein (MBP) regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by GFP reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. My PhD project is to study the effect of demyelination and remyelination in Xenopus Laevis in this transgenic line. We wish to answer two questions using this transgenic. First, the origin of remyelinating cells that replace the ablated oligodendrocytes. We have shown that, Sox10+ OPCs, which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination. The second question is to examine the effect of demyelination of retinal ganglion cell (RGC) axonal arbor morphology. We developed an experimental set up to image the RGC arbor morphology in an awake stage 55 tadpole in real time. We show that the arbor is more sensitive than the control to imaging but there is no change in motility of the arbor
Contribution à l'étude de la démyélinisation et la remyélinisation chez Xenopus
We have generated a Xenopus laevis transgenic line, pMBP-eGFP-NTR, allowing conditional ablation of myelin-forming oligodendrocytes. In this transgenic line the transgene is driven by the proximal portion of myelin basic protein (MBP) regulatory sequence, specific to mature oligodendrocytes. The transgene protein is formed by GFP reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. My PhD project is to study the effect of demyelination and remyelination in Xenopus Laevis in this transgenic line. We wish to answer two questions using this transgenic. First, the origin of remyelinating cells that replace the ablated oligodendrocytes. We have shown that, Sox10+ OPCs, which are already present in the optic nerve prior to the experimentally induced demyelination, are responsible for remyelination. The second question is to examine the effect of demyelination of retinal ganglion cell (RGC) axonal arbor morphology. We developed an experimental set up to image the RGC arbor morphology in an awake stage 55 tadpole in real time. We show that the arbor is more sensitive than the control to imaging but there is no change in motility of the arbor.La lignĂ©e transgĂ©nique pMBP-eGFP-NTR, que nous avons gĂ©nĂ©rĂ©e chez Xenopus laevis, permet une ablation conditionnelle des oligodendrocytes myĂ©linisants, dont la consĂ©quence est une dĂ©myĂ©linisation. Dans cette lignĂ©e transgĂ©nique, le transgĂšne est formĂ© par une protĂ©ine de fusion entre le rapporteur GFP (Green Fluorescent Protein= protĂ©ine fluoresçant en vert) et une enzyme de sĂ©lection, la nitrorĂ©ductase dâE. Coli. Cette enzyme, a la propriĂ©tĂ© de rĂ©duire le radical nitrite (NO2) de certains substrats (comme le mĂ©tronidazole) en dĂ©rivĂ© hydroxylamine extrĂȘmement toxique pour la cellule qui lâexprime. Lâexpression de ce transgĂšne est contrĂŽlĂ©e par la portion proximale du gĂšne MBP (myelin basic protein), sĂ©quence rĂ©gulatrice, dont lâĂ©quipe avait dĂ©montrĂ©, chez la souris, quâelle ne sâexprime que dans les oligodendrocytes myĂ©linisants, ce qui câest vĂ©rifiĂ© chez le xĂ©nope. Mon projet se proposait dâĂ©tudier les consĂ©quences de la dĂ©myĂ©linisation et de la remyĂ©linisation dans cette lignĂ©e transgĂ©nique de Xenopus laevis. Mon objectif avait pour but de rĂ©pondre Ă deux questions; Tout dâabord, quâelle est la nature des cellules qui remplacent les oligodendrocytes Ă©liminĂ©s: Nous montrons que les cellules responsables de la remyĂ©linisation sont les prĂ©curseurs des oligodendrocytes (OPCs), cellules GFP nĂ©gatives caractĂ©risĂ©es par lâexpression du facteur de transcription Sox10. Ces cellules OPCs sont dĂ©jĂ prĂ©sentent dans le nerf optique avant lâĂ©vĂ©nement de dĂ©myĂ©linisation. La seconde question visait Ă examiner les consĂ©quences dâune dĂ©myĂ©linisation sur lâarborisation des axones des cellules ganglionnaires de la rĂ©tine. A cette fin nous avons mis au point un outil expĂ©rimental permettant de visualiser lâarborisation des projections tectales des axones des cellules ganglionnaires de la rĂ©tine par microscopie in vivo rĂ©alisĂ©e sur le tĂȘtard au stade 55. Nous montrons que bien que cette arborisation soit plus sensible Ă lâimagerie aprĂšs dĂ©myĂ©linisation, chez le transgĂ©nique que chez le contrĂŽle, cela nâentraine pas de changement de la motilitĂ© de lâarborisation
Intrinsic membrane properties of locus coeruleus neurons in Mecp2-null mice
Rett syndrome caused by mutations in methyl-CpG-binding protein 2 (Mecp2) gene shows abnormalities in autonomic functions in which brain stem norepinephrinergic systems play an important role. Here we present systematic comparisons of intrinsic membrane properties of locus coeruleus (LC) neurons between Mecp2â/Y and wild-type (WT) mice. Whole cell current clamp was performed in brain slices of 3- to 4-wk-old mice. Mecp2â/Y neurons showed stronger inward rectification and had shorter time constant than WT cells. The former was likely due to overexpression of inward rectifier K+ (Kir)4.1 channel, and the latter was attributable to the smaller cell surface area. The action potential duration was prolonged in Mecp2â/Y cells with an extended rise time. This was associated with a significant reduction in the voltage-activated Na+ current density. After action potentials, >60% Mecp2â/Y neurons displayed fast and medium afterhyperpolarizations (fAHP and mAHP), while nearly 90% WT neurons showed only mAHP. The mAHP amplitude was smaller in Mecp2â/Y neurons. The firing frequency was higher in neurons with mAHP, and the frequency variation was greater in cells with both fAHP and mAHP in Mecp2â/Y mice. Small but significant differences in spike frequency adaptation and delayed excitation were found in Mecp2â/Y neurons. These results indicate that there are several electrophysiological abnormalities in LC neurons of Mecp2â/Y mice, which may contribute to the dysfunction of the norepinephrine system in Rett syndrome
Loss of PRMT1 in the CNS induces reactive astrocytes and microglia during postnatal brain development
Cryogel scaffolds for regionally constrained delivery of lysophosphatidylcholine to central nervous system slice cultures: a model of focal demyelination for multiple sclerosis research
The pathology of multiple sclerosis (MS) is typified by focal demyelinated areas of the brain and spinal cord, which results in axonal degeneration and atrophy. Although the field has made much progress in developing immunomodulatory therapies to reduce the occurrence of these focal lesions, there is a conspicuous lack of licensed effective therapies to reduce axonal degeneration or promote repair. Remyelination, carried out by oligodendrocytes, does occur in MS, and is protective against axonal degeneration. Unfortunately, remyelination is not very efficient, and ultimately fails and so there is a research focus to generate new therapeutics to enhance remyelination leading to neuroprotection.
To develop these therapies, we need preclinical models that well reflect remyelination in MS. We have previously characterized an ex vivo model that uses lysophosphatidylcholine (LPC) to cause acute and global demyelination of tissue slices, followed by spontaneous remyelination, which has been widely used as a surrogate for in vivo rodent models of demyelination. However, this ex vivo model lacks the focal demyelinated lesions seen in MS, surrounded by normal tissue from which the repairing oligodendrocytes are derived. Therefore, to improve the model, we have developed and characterized small macroporous cryogel scaffolds for controlled/regional delivery of LPC with diameters of either 0.5, 1 or 2âŻmm. Placement of LPC loaded scaffolds adjacent to ex vivo cultured mouse brain and spinal cord slices induced focal areas of demyelination in proximity to the scaffold. To the best of our knowledge, this is the first such report of spatial mimicry of the in vivo condition in ex vivo tissue culture. This will allow not only the investigation into focal lesions, but also provides a better platform technology with which to test remyelination-promoting therapeutics