Bin JM and Lyons DA (2016), Imaging Myelination In Vivo Using Transparent A...

XB-ART-54919

Brain Plast 2016 Dec 21;21:3-29. doi: 10.3233/BPL-160029.

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Imaging Myelination In Vivo Using Transparent Animal Models.

Bin JM , Lyons DA .

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Myelination by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system is essential for nervous system function and health. Despite its importance, we have a relatively poor understanding of the molecular and cellular mechanisms that regulate myelination in the living animal, particularly in the CNS. This is partly due to the fact that myelination commences around birth in mammals, by which time the CNS is complex and largely inaccessible, and thus very difficult to image live in its intact form. As a consequence, in recent years much effort has been invested in the use of smaller, simpler, transparent model organisms to investigate mechanisms of myelination in vivo. Although the majority of such studies have employed zebrafish, the Xenopus tadpole also represents an important complementary system with advantages for investigating myelin biology in vivo. Here we review how the natural features of zebrafish embryos and larvae and Xenopus tadpoles make them ideal systems for experimentally interrogating myelination by live imaging. We outline common transgenic technologies used to generate zebrafish and Xenopus that express fluorescent reporters, which can be used to image myelination. We also provide an extensive overview of the imaging modalities most commonly employed to date to image the nervous system in these transparent systems, and also emerging technologies that we anticipate will become widely used in studies of zebrafish and Xenopus myelination in the near future.

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Genes referenced: gpi lgals4.2 mbp sox10

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	Fig.1. Timeline of developmental myelination in zebrafish larvae and Xenopus tadpoles. Schematic indicates rapid development of zebrafish and Xenopus embryos from one cell stage (left) to larvae (right) with myelinated axons. hpf = hours post fertilisation. dpf = days post fertilisation.
	Fig.2. Generation of transgenic zebrafish to visualize myelinating oligodendrocytes. A) Transgenic animals with mosaic fluorescent reporter expression in myelinating oligodendrocytes can be generated by injection of plasmid at the one cell stage, and visualized in the injected animals at early larval stages. B) Transgenic animals with stable expression of a transgene that labels all myelin in the animal are first injected with plasmid as in A, then grown to adulthood. Adults are bred with non-transgenic animals to identify those in which plasmid integration has occurred in the germ-line and which can generate offspring with expression in all myelinating oligodendrocytes.
	Fig.3. Example strategies to control gene expression in zebrafish and Xenopus. A) Plasmids that can drive expression of the transcriptional activator Gal4-VP16 can be co-injected into embryos together with plasmids that encode genes-of-interest downstream of Upstream Activator Sequences (UAS). Gal4 trans-activates UAS and can amplify gene expression. When injected in this way, it leads to mosaic gene expression. Stable transgenic lines expressing Gal4 in specific cell types or UAS effectors can be generated as in Fig. 1. B) The Gal4-UAS system can be modified to control expression of multiple genes from one plasmid. In the Janus system Gal4 trans-activates UAS, which drives gene expression in both directions, if minimal promoters are present upstream of the genes of interest. C) Alternative strategies exist to express two genes from one construct including the self-cleaving 2A peptide sequence, which can be placed between two open reading frames in one mRNA, which is then translated as two polypeptides due to ribosome skipping. D) Temporal control of gene expression can be mediated by the use of heat sensitive promoters, which are only active at specific temperatures.
	Fig.4. Time-lapse microscopy reveals dynamic behavior of oligodendrocytes. A and B show images from a time-lapse made on a spinning disk confocal microscope, using the stable zebrafish transgenic line Tg(nkx2.2a:meGFP) as a reporter, exemplifying the rapid dynamics of oligodendrocyte processes. Scale bars = 5 μm. A shows a cell in the process of initiating myelination. Arrowhead points to an elongated membrane rich process that may represent at nascent myelin sheath that remains stable for the frames shown, whereas the arrow points to a similar elaboration of membrane that is resorbed during the same short period. B shows an oligodendrocyte with a process (arrowhead) that acutely transitions in morphology from a profile indicative of a nascent myelin sheath (0’ and 8’) to that of an exploratory process (4’, 12’, 16’ and 20’).
	Fig.5. Confocal super-resolution imaging. A+B are images of the stable transgenic reporter mbp:eGFP-CAAX, which labels myelin sheaths, taken in confocal (A) and Airyscan (B) modes. A clear improvement in resolution in the X-Y plan is observed. C indicates improvement in z-axis resolution in Airyscan mode. The red line indicates the position where the Y-Z projections on the right were taken. Definitive circular rings of myelin membrane are observable in the Airyscan mode. Scale bars = 5 μm.
	Fig.6. Transgenic tool to visualize myelin along individual axons in vivo. A fluorescent fusion of GFP with the GPI anchored axonal protein contactin1a can be expressed in individual neurons together with a fluorescent protein that labels the entire cell, e.g. Tandem dimer (Td) Tomato (A). Gaps in GFP-Cntn1a localisation along axons (A+B) correspond to the locations of myelin sheaths, as shown in (C) where all myelin is labelled using the sox10:mRFP reporter. Scale bars: A = 20 μm, B+C = 5 μm.

References [+] :

Ackerman, The scales and tales of myelination: using zebrafish and mouse to study myelinating glia. 2016, Pubmed