Figure 1.
Static images of myelination from mouse cultures in vitro using differentiation markers.
A–B) Confocal image acquired using FV1000 with 60× 0.75NA (A–C) showing the initial contact between oligodendrocytes (O4, red) and neurites (SMI-31, green) in a wild type mouse myelinating culture. After DIV 9 the oligodendrocyte's processes appear to align along neurites B). Subsequent stages in axon/oligodendrocyte contact and wrapping where PLP/DM20 can be detected alongside expression of the O4 antibody. The staining suggests that myelin sheaths wrap around segments of the axon at this stage (solid regions of red and green; arrows) around DIV 9. C) Image of a culture after 28 days in vitro. The oligodendrocyte's many processes appear to “fill-out” (solid green sheaths) from the initial spirals (arrows) of the membrane visualised by PLP/DM20 (green) around SMI-31 (red) neurites. D) Single contiguous myelin sheaths begin to appear around 17–18 days in vitro using anti-PLP/DM20. Nodes of Ranvier are apparent between the internodes of myelin (asterisk) using an antibody to the nodal protein Caspr (insert). Image taken with and a Zeiss Axioplan II, 20×, 0.75NA (D) and similar images were observed from 5–10 separate experiments.
Figure 2.
Cyto-GFP labelled neurospheres generate oligodendrocytes which spiral cell process around neuritis.
A) Neurospheres expressing cyto-GFP under the β-actin promoter were added on day 6 to myelinating cultures prepared from wild type mouse embryos. Confocal imaging (Zeiss 710, 63×, 1.4NA) on DIV 21 demonstrates that cell processes initially form spirals around neurites. Since cyto-GFP (green) is only present in the cytoplasm we co-labelled with the O4 antibody which labels myelin membrane to determine the membrane and cyto-GFP are contiguous. It can be seen that at some point the GFP and O4 immunoreactivity are contiguous over the neurite (SMI-31, blue, arrowheads). B) Staining with anti-PLP/DM20 (red) and anti-GFP shows that myelin membrane lacking cyto-GFP extends from the cyto-GFP strands. C) Many cytoplasmic cuffs of cyto-GFP labelled with anti-PLP/DM20 can be seen along the neurite (arrows, blue). Representative images from at least 3 separate experiments.
Figure 3.
In vitro time-lapse imaging of cyto-GFP labelled OPC-like cells in shiverer myelinating cultures reveal dynamic cellular interactions.
Neurospheres expressing cyto-GFP under the β-actin promoter were added to myelinating cultures prepared from shiverer embryos on DIV 13 and visualised on DIV 17 over 24 hours in 4 min intervals, using a Nikon TE2000 time-lapse microscope (40×, 0.75NA short distance working lens) with perfect focus. A–I) Images captured from a time-lapse sequence illustrate cells with morphology typical of oligodendrocyte progenitor cells (OPCs). Spirals of processes appear over the neurites. Both the cell soma and processes are highly motile, continually moving along the neurites. Processes appear to move over the nerve bundles (arrows and asterisk). J) Manual tracking of the cell bodies of the entire video illustrates the pathway of the putative OPC-like cells (asterisks green and blue) over time, demonstrating their highly motile behaviour. K) A sister culture was immunostained for myelin (PLP/DM20; green) and neurites/axons (SMI-31; red) on DIV 27, demonstrating that the added cyto-GFP neurospheres myelinated the axons. See Video S1. Representative images from at least 2 separate experiments.
Figure 4.
Time-lapse imaging of fluorescently labelled cells in association with neurites in vitro.
A–E) Myelinating cultures generated from a mix of wild type/beta-actin mice were visualised using time-lapse microscopy (Nikon TE2000 (60×, 0.75NA) over 16 hr in 5 min intervals on DIV 27 after the addition of wild type neurospheres previously infected with lentivirus carrying dsRed/GFP gene and addition of cyto-GFP cells. Two cell types were followed over time, one that expressed DS red/cyto-GFP and the other cyto-GFP. A–E) Strongly positive green cells typical of cyto-GFP morphologically resembled oligodendrocytes in contact with neurite bundles. The membrane appears to ruffle and form flaps/bubbles (arrow). In addition, the soma changes its location with respect to the neurite processes, over time, by moving closer to the neurite bundle. C–E) Dynamic imaging over 7.5 hours of a dsred/GFP labelled cell (asterisk) which was engulfed by a cell resembling a microglial cell (yellow arrow). This fluorescence was very much weaker than the cells generated from the beta-actin cyto-GFP mouse. Time frames obtained with 40× magnification (long distance working lens) and without perfect focus. See Video S2. F) Immunostaining of a similar culture around the same time confirmed MBP expression. Representative video from 1 experiment, seen in duplicate.
Figure 5.
Evidence that oligodendrocytes form spiral processes around neurites.
A–C) Confocal image acquired using a Zeiss 710, (63×, 1.4NA) with maximum projection of a z stack, with step interval of 0.06 µm of a wild type culture with exogenous added cyto-GFP labelled neurospheres, immunolabelled on 27 DIV using anti-GFP and anti-PLP/DM20. Cyto-GFP strands were visualised to spiral around an neurite on top of intense PLP/DM20 immunoreactivity. D–H) To confirm that the process is contiguous around the neurite, we took serial confocal images of a z stack with a step size of 0.24 µm in depth, of a cyto-GFP (green) strand spirally looping around a myelinated axon (MBP, red) I) The pattern of axonal wrapping is summarised in the adjacent schematic where D–H represent the images in Fig. 5D–H) visualised in cross section and longitudinally. Ji–ii) Illustrates a schematic of the myelinated fibre and the cyto-GFP immunoreactivity. Ji) Illustrates the typical schematic of a myelinated fibre with the lateral cytoplasmic loop forming a straight line. In our images it appears that this may not be the case and that it forms a spiral over the axon, at least during wrapping. Representative images from at least 5 separate experiments.
Figure 6.
Time-lapse imaging of the putative assembly of myelin membrane.
A) Neurospheres expressing farns-GFP were added to shiverer myelinating cultures on DIV 19 and time-lapse imaging (Nikon TE2000) was performed on 29 DIV, over 24 hr with 4 min time intervals. Ai–iv) Magnified view of the inset in A illustrates a farns-GFP process looping around a presumptive neurite and forming a membranous protrusions or ‘bubble’ (yellow arrow head). This membrane bubble appears to moves along the neurite over time. See Video S3. B) The cells from the Petri dish imaged with confocal microscopy were immunostained with anti-GFP and anti-MBP to confirm differentiation of cyto-GFP labelled oligodendrocytes.C–F) Time-lapse sequence of the same culture for a period of 30 hours, with 3 min time interval, on 24 DIV revealed membrane cuffs (arrowheads) extending and joining up over a neurite. After about 13 hours, the farns-GFP-positive cuffs were observed to form a single, united thick membrane sheath over a neurite. G) Manual tracking of the pathway of a weakly GFP-positive cell which was possibly associated with the membranous fragments. See Video S4. Representative images of membranous bubbles from at least 4 separate videos and membrane cuffs seen in 1 video but in at least 3 static images.
Figure 7.
Time-lapse imaging of the elongation of a putative farns-GFP labelled myelin-like sheath.
Neurospheres expressing farns-GFP were added to shiverer myelinating cultures on 19 DIV and time-lapse imaging (Nikon TE2000) performed on 26 DIV, for a period of 14 hr with 4 min time interval. Ai–iii) A farns-GFP process increases in length by 10 µm, over the time course. Membranous protrusions were seen (yellow arrowhead) in addition to the cell body of the farns-GFP labelled oligodendrocyte-like cell (yellow arrow). B) MBP staining of cells in the Petri dish using a Zeiss 710 (×63, 1.4NA) after imaging confirms that farns-GFP expressing cells belong to the oligodendroglial lineage. See Video S5. Representative images from at least 5 separate experiments.
Figure 8.
Immunohistochemistry of transplanted neurospheres demonstrate that cyto-GFP labelled cells form early and mature myelinating oligodendrocytes.
Cyto-GFP-expressing neurospheres were transplanted into a shiverer mouse 3, 7 or 15 days post-transplantation, and 10 µm thick frozen sections were cut and immunolabelled with antibodies to GFP and MBP. Low magnification image of a dorso-vental section of spinal cord, 15 days post-transplantation showing GFP (A) and MBP (B) immunostaining. Transplanted cells were located in both grey and white matter (dorsal columns are delineated by the dotted lines) and expressed MBP-positive myelin sheaths. C) A pre-myelinating cell in which multiple fine GFP positive processes emanate from a central cell body. The soma is also lightly stained with MBP, confirming the identity of the cell as that of the oligodendroglial lineage. MBP-positive myelin sheaths, belonging to a second cell are seen in the bottom left hand corner of the images. D) An early myelinating cell in which short MBP-positive profiles are present at the periphery of the GFP-positive soma. All images were obtained using epifluorescence Olympus microscope (FV10 ASW). Representative images from at least 30 separate experiments.
Figure 9.
Confocal images of transplanted cyto-GFP expressing cells in the shiverer spinal cord.
Four weeks after transplantation of cyto-GFP expressing neurospheres, fixed sections of the shiverer spinal cord were immunolabelled for MBP (red) and GFP (green). A) A cyto-GFP labelled cell appears to extend spirals of cytoplasm around an MBP-positive myelin-like sheath (yellow arrows). Below the cell body, cyto-GFP is seen at the lateral edges (in relation to the long axis of the sheaths, white arrow) of adjacent sheaths and probably represents the cytoplasm filled paranodal loops on either side of the node of Ranvier (asterisks). B) Schematic of visualisation of the sections in C and D. Ci–ii and Di–ii) Spiral of GFP cytoplasm was followed by focussing up and down through the plane of view where they crossed up, traversed the axonal surface, then crossed down again representing the looping as shown in the schematic in B. E–G) 3D reconstruction of cyto-GFP structures (E), illustrates cyto-GFP either side of a space typical of a node of Ranvier (white arrows). F) is a tilted perspective of E) and shows the cyto-GFP form complete rings (white arrows representing the same position in E), consistent with the morphology of paranodal loops. I) Asymmetric caspr positive structures in association with cyto-GFP, at either side of a heminode. On the left, caspr forms a single vertical line and co-localises with cyto-GFP from the myelinating cell. On the right, caspr appears like a loose coil, consistent with its pattern of expression in non-myelinated axons. All images were acquired using an Olympus FV1000 confocal microscope (×60, 1.35NA). Representative images from at least 10 separate experiments.
Figure 10.
Ex vivo imaging of transplanted cyto-GFP and farns-GFP labelled neurospheres into the shiverer and Thy1-CFP*shi/shi mouse demonstrate extension of cells processes.
A) Using multiphoton microscopy the morphology of the cyto-GFP-transplanted cells in the fixed sections of the spinal cord parenchyma was first visualised in serial images with 0.1 µm step size. Second harmonic generation (SHG) blue signal was probably generated by collagen. See Video S6. B) Image acquired using a Zeiss 7 MP of a cyto-GFP positive cell 10 days after transplantation into the non-fixed ex vivo spinal cord that resembles a typical process bearing oligodendrocyte. C) Eight days post-transplantation cytoplasmic GFP-positive cells were seen to extend processes making contacts with the axons at several points (white arrows). D) Nine days post-transplantation farnesylated GFP-labelled cells were imaged to align with the CFP-labelled axons extending thick dense processes (white arrow). B and D acquired using Zeiss 7 MP microscope (×20, 0.95NA). A and C acquired using the LaVision BioTec TRIM scope, (20×, 0.95NA). Representative images from at least 3 separate experiments.
Figure 11.
Ex vivo imaging of axon-glial interactions after transplantation of cyto-GFP and farns-GFP labelled cells into a Thy1-CFP*shi/shi mouse.
A) Cyto-GFP-expressing neurospheres were transplanted into a shiverer mouse expressing CFP under the Thy1 promoter, Thy1-CFP*shi/shi. Z stacks at 1 µm intervals were acquired with the multiphoton microscopy of an ex vivo spinal cord at day 15 after transplantation, and presented as a volume projection, over a time course of 20 min. Cyto-GFP-positive processes extending from a cell body can be seen to align with the CFP-positive axons. Z interval size was 1 µm. GFP and CFP were excited simultaneously at 860 nm. Bi–iii) Smoothed illustration after applying blend configuration in Imaris imaging software of the co-localization of GFP positive processes with CFP positive axons acquired using time-lapse microscopy. It can be seen that the GFP-positive cell process appears to extend over areas overlying axons. See Video S7. C–F) ex vivo imaging of farns-GFP-labelled cells and CFP-positive axons 8 days post-transplantation. (A–B acquired using the LaVision TRIM microscope, 20×. 0.95NA) C) farns-GFP-transplanted cells (asterisk) were imaged to extend flatten processes, making contact with the CFP-positive axons (dotted square), 8 days post-transplantation into Thy1-CFP*shi/shi mouse. In the same field of view, a neuronal body (purple arrow) was also imaged. D,E) Time-lapse imaging over a z-stack, with 1 µm step size, illustrating the generation and extension of a farns-GFP-positive cell process with the formation of new membrane bubble (arrow, see Video S8). F) farns-GFP processes were generally thick and formed membrane protrusions (arrow). C–F images were acquired using Zeiss 7 MP (20×, 0.95NA). Representative video (A,B) from 1 experiment but static images taken from at least 5 separate experiments. C–F illustrates representative images from at least 3 separate experiments.