Multicolor analysis of oligodendrocyte morphology, interactions, and development with Brainbow

Abstract

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single-out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre-lox recombination, previously used for visualizing axons and neurons. We used tamoxifen-inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single-cell resolution.

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve

“…To perform our analyses, we first needed to determine the theoretical number of axons that an OL can reach, N A . Analysis of photomicrographs published by Dumas et al [7] reveals that the maximum length of the primary process of an OL in a mouse optic nerve is ~30 μm, which we take as the radius of influence of an OL. Given that axonal density in the mouse optic nerve is approximately one axon per μm 2 [11–13], we conclude that each primary process of a single OL could theoretically reach N A = 2800 axons.…”

“…Secondly, Dumas and her colleagues [7] found that adjacent OLs were often observed to form juxtaposed myelin internodes on the same axon i.e. share a common set of axons (we will refer to this finding as ‘Observation B’).…”

“…To investigate this question, we examined two sets of quantitative data published in 2015 by Dumas et al [7], who analyzed the topographic organization of myelin internodes from clonally labeled OLs in the postnatal mouse optic nerve, a white matter tract in which almost the entire length of every axon is myelinated [8–10]. The morphology of individual OLs was visualized by inducing the expression of different combinations of fluorescent reporter proteins in OLs in a stochastic manner that relied upon low dose administration of tamoxifen to PLP : CreER T2 ;CAGbow transgenic mice.…”

Extraocular Source of Oligodendrocytes Contribute to Retinal Myelination and Optokinetic Responses in Zebrafish

“…The behaviors of OLs in zebrafish retinas are similar to the behaviors of OLs in the optic nerves of mice. 8,9 The lengths and number of internodes made by each OL varied greatly in different CNS regions. 38 We found that, in comparison with the spinal cord, 32,33,37 retinas tend to have longer and a greater number of internodes.…”

“…6,7 Oligodendrocyte precursors continue to proliferate and generate myelinating OLs into adulthood, and late-born OLs generate many more internodes, much shorter in length, than early-born OLs in mice optic nerves. 8,9 It is important to identify whether the OLs at different ages in retinas may have different behaviors regarding the number and lengths of internodes.…”