Kaya F , Mannioui A , Chesneau A , Sekizar S , Maillard E , Ballagny C , Houel-Renault L , Dupasquier D , Bronchain O , Holtzmann I , Desmazieres A , Thomas JL , Demeneix BA , Brophy PJ , Zalc B , Mazabraud A .

089000 Wellcome Trust , WT089000 Wellcome Trust , Wellcome Trust

             central nervous system myelination

	Figure 1. Structure and expression of the pMBP-eGFP-NTR transgene. A, Schematic diagram of the pMBP-eGFP-NTR construct. The transgene contains the eGFP open reading frame fused to that of E. coli NTR placed under the control of the DNA regulatory sequence of the murine MBP gene (-1907 bp and +36 bp). B, RT-PCR performed on RNA extracted from brains of transgenic (TG) or wild-type (WT) tadpoles used to amplify a 394 bp fragment corresponding to the junction of eGFP/NTR sequence. C–F, Transgene expression as assessed by GFP fluorescence (C–E) or immunolabeling (F ) in a pMBP-eGFP-NTR transgenic tadpole at stage 55. Dorsal view of the head (C) and sagittal view of the tail (D). Expression is only observed in the CNS (brain and spinal cord) but not in the peripheral nervous system. Inset in C is a higher magnification to illustrate the detection of GFP in the optic nerve. E, In vivo stack of images of the optic nerve obtained by two-photon microscopy. Note the fluorescent processes of the GFP + cells. F, Confocal image of a whole mount of the optic chiasm immunostained for MBP (red) and GFP (green). Note the GFP + cell bodies extending their processes toward the strongly MBP +myelinated fibers. Scale bar (in E) C, D, 2 mm; E, F, 50 um; inset (C), 1 mm.
	Movie 1 (still from). Faint detection of GFP in myelin along optic nerve axons of stage 50 pMBP-eGFP- NTR transgenic tadpole. At this early developmental stage, the wrapping process is ongoing, therefore myelin is still not completely compact and this allows diffusion of GFP in the myelin cytoplasmic compartment. This movie represents the Z-projection of 17 successive optical sec- tions across the thickness of the optic nerve imaged by two-photon microscopy.
	Figure 2. The pMBP-eGFP-NTR transgene drives expression in mature oligodendrocytes. Coronal tissue sections across the medulla of pMBP-eGFP-NTR tadpole at stage 55 co-immunostained for GFP and successive markers of different cells types. A–C, GFP and APC, a specific marker of mature oligodendrocytes. Note the complete overlap of GFP labeling with that of APC. D–F, GFP and Nkx2.2, a marker of progenitor of oligodendrocytes and mature oligodendrocytes. Note that cells doubly labeled for GFP and Nkx2.2 are localized in the white matter tract, with no GFP detection in Nkx2.2 􏰇 progenitors in the ventral ventricular layer (white arrows in F ). G–I, GFP and GFAP, a marker of astrocytes. J–L, GFP and Hu, a pan-neuronal marker. Note the complete exclusion of GFP labeling with either GFAP (I ) or Hu (L). Scale bar (in C) A–F, J–L, 50 um; G–I, 25 um.
	Figure 3. Oligodendrocyte depletion and apoptosis in transgenic tadpoles following MTZ treatment. A,B, Whole-mount two-photon microscopy of the optic nerve at stage 55 showing GFP+cells in transgenic animals untreated (A) or MTZ-treated for 3 d (B). MTZ induced a severe depletion of GFP+ cells in the optic nerve. C–H, Immunolabeling for GFP and activated caspase3 (Casp3) on coronal sections across the medulla in untreated (C, E, G) or MTZ-treated (D, F, H ) transgenic animals. In MTZ-treated animals, activated caspase3 is detected in GFP + cells. Scale bar (in G) A, B, 150um; C–H, 50 um.
	Figure 4. MTZ treatment induces demyelination without axonal damage. A, B, Myelinated fibers staining with Luxol fast blue on coronal sections across the medulla at stage 55 in untreated (A) or MTZ-treated (B) transgenic animals. C–H, Whole mount of optic nerve at stage 55 immunostained for neurofascin (green) and SMI-31 (red) in untreated (C–E) or MTZ-treated (F–H ) transgenic animals. Insets in C and F are high magnifications of nodes of Ranvier showing a strong signal for NF 155 in the paranodal domain and a weaker signal in the node (NF186) in a control animal (C), where in case of partial demyelination, only hemiparanodes are labeled (F ). MTZ treatment induced demyelination, characterized by disorganization of the neurofascin 􏰇 nodes of Ranvier (F, H ) with numerous heminodes compared with control (C, E). In MTZ-treated tadpoles, SMI-31 axons had a normal appearance (G, compared with D), suggesting that MTZ-induced demyelination does not affect the axons. Scale bar (in F ) A,B,17 um; C–H, 5um;insets (C,F), 2um.

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