Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate
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Evaluation Summary:
Almeida and Macklin provide one of the first studies to closely examine early oligodendrocyte behaviors at high resolution. These studies use live imaging in zebrafish to provide valuable new insights about the earliest onset of myelination in the central nervous system and add to a body of work showing how oligodendrocytes initiate and maintain myelin sheaths.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Oligodendrocytes in the central nervous system exhibit significant variability in the number of myelin sheaths that are supported by each cell, ranging from 1 to 50 (1-8). Myelin production during development is dynamic and involves both sheath formation and loss (3, 9-13). However, how these parameters are balanced to generate this heterogeneity in sheath number has not been thoroughly investigated. To explore this question, we combined extensive time-lapse and longitudinal imaging of oligodendrocytes in the developing zebrafish spinal cord to quantify sheath initiation and loss. Surprisingly, we found that oligodendrocytes repetitively ensheathed the same axons multiple times before any stable sheaths were formed. Importantly, this repetitive ensheathment was independent of neuronal activity. At the level of individual oligodendrocytes, each cell initiated a highly variable number of total ensheathments. However, ~80–90% of these ensheathments always disappeared, an unexpectedly high, but consistent rate of loss. The dynamics of this process indicated rapid membrane turnover as ensheathments were formed and lost repetitively on each axon. To better understand how these sheath initiation dynamics contribute to sheath accumulation and stabilization, we disrupted membrane recycling by expressing a dominant-negative mutant form of Rab5. Oligodendrocytes over-expressing this mutant did not show a change in early sheath initiation dynamics but did lose a higher percentage of ensheathments in the later stabilization phase. Overall, oligodendrocyte sheath number is heterogeneous because each cell repetitively initiates a variable number of total ensheathments that are resolved through a consistent stabilization rate.
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Evaluation Summary:
Almeida and Macklin provide one of the first studies to closely examine early oligodendrocyte behaviors at high resolution. These studies use live imaging in zebrafish to provide valuable new insights about the earliest onset of myelination in the central nervous system and add to a body of work showing how oligodendrocytes initiate and maintain myelin sheaths.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Our understanding of the early stages of myelination within the CNS is relatively rudimentary. In this manuscript the authors use selective cell labeling to visualize the initial interactions between individual oligodendrocytes and their target axons in the developing zebra fish with the goal of understanding the regulation of myelin sheath formation.
There are considerable strengths to the manuscript. The work extends earlier studies through the use of high spatial and temporal resolution analysis. This approach reveals a highly dynamic interaction between oligodendrocyte processes and local axons that had not previously been appreciated. The data on the initial interactions between an individual oligodendrocyte and its target axons is closely analyzed, which reveals a number of interesting traits. For …
Reviewer #1 (Public Review):
Our understanding of the early stages of myelination within the CNS is relatively rudimentary. In this manuscript the authors use selective cell labeling to visualize the initial interactions between individual oligodendrocytes and their target axons in the developing zebra fish with the goal of understanding the regulation of myelin sheath formation.
There are considerable strengths to the manuscript. The work extends earlier studies through the use of high spatial and temporal resolution analysis. This approach reveals a highly dynamic interaction between oligodendrocyte processes and local axons that had not previously been appreciated. The data on the initial interactions between an individual oligodendrocyte and its target axons is closely analyzed, which reveals a number of interesting traits. For example, while dorsal cells have a higher number of initial axonal interactions and ultimately myelinate more axons than ventral cells, the proportion of initial interactions that lead to a myelin sheath is similar between the two populations. To begin to examine the molecular regulation of the initial oligodendrocyte and axon interactions and subsequent formation of myelin sheaths the authors perturb selective components of the endocytic pathway and provide evidence that disruption of Rab5 selectively affects the long-term stabilization of myelin sheaths.
While there are some new advances in the current manuscript, the significance of many of the observations is unclear. For example, while the data documents extensive interactions between oligodendrocytes and axons, the nature of those interactions is not well defined. The authors describe the loss of olig/axon interactions as "ensheathment destabilization" however, it is not clear from the data that they don't represent simple oligodendrocyte process retraction.
The different interactions of dorsal and ventral cells with their target axons is interesting and may reflect different oligodendrocyte populations or environments.
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Reviewer #2 (Public Review):
The study by Almeida and Macklin takes advantage of the power of zebrafish as a model system to study the dynamic behaviour of myelinating oligodendrocytes in a living system. The study provides valuable new insights into how oligodendrocytes undertake myelination and will be of use to the field. One particularly interesting finding of the study is the observation that individual myelinating processes tend to iteratively wrap and unwrap their early ensheathments from axons numerous times before fully committing to myelinating that axon, or fully withdrawing from the axon. This occurs at a much higher rate than one might have expected and will be interesting to disentangle in the future. The authors also find use genetic perturbations in myelinating cells of the zebrafish to disrupt the endocytic pathway and …
Reviewer #2 (Public Review):
The study by Almeida and Macklin takes advantage of the power of zebrafish as a model system to study the dynamic behaviour of myelinating oligodendrocytes in a living system. The study provides valuable new insights into how oligodendrocytes undertake myelination and will be of use to the field. One particularly interesting finding of the study is the observation that individual myelinating processes tend to iteratively wrap and unwrap their early ensheathments from axons numerous times before fully committing to myelinating that axon, or fully withdrawing from the axon. This occurs at a much higher rate than one might have expected and will be interesting to disentangle in the future. The authors also find use genetic perturbations in myelinating cells of the zebrafish to disrupt the endocytic pathway and find that interfering with endocytosis reduces the number of myelin sheaths that individual oligodendrocytes make. The strengths of the manuscript are in the use of zebrafish as a model and the careful analysis of time-lapse movies of myelination by oligodendrocytes. Further work will be required to understand the mechanisms by which the early myelinating processes wrap and unwrap their sheaths from axons, what commits them to final fate of staying on an axon and leaving and precisely what role endocytic pathway modulation has on myelination.
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Reviewer #3 (Public Review):
Almedia and Macklin sought to characterize oligodendrocyte behavior at the earliest onset of myelination in the central nervous system. By sparsely labeling oligodendrocytes with transgenic fluorescent reporters in zebrafish larvae, they show that oligodendrocytes in the dorsal and ventral spinal cord have characteristic numbers of sheaths and sheath lengths. With impressive and technically laborious time-lapse imaging, they demonstrate that oligodendrocytes repetitively sample axon segments before stabilizing a nascent sheath, with most (~90%) immature sheaths failing to stabilize. They quantify differences in dorsal and ventral oligodendrocyte sampling and convincingly show that dorsal oligodendrocytes form more sheaths due to increased sampling relative to ventral, with similar rates of sheath retraction. …
Reviewer #3 (Public Review):
Almedia and Macklin sought to characterize oligodendrocyte behavior at the earliest onset of myelination in the central nervous system. By sparsely labeling oligodendrocytes with transgenic fluorescent reporters in zebrafish larvae, they show that oligodendrocytes in the dorsal and ventral spinal cord have characteristic numbers of sheaths and sheath lengths. With impressive and technically laborious time-lapse imaging, they demonstrate that oligodendrocytes repetitively sample axon segments before stabilizing a nascent sheath, with most (~90%) immature sheaths failing to stabilize. They quantify differences in dorsal and ventral oligodendrocyte sampling and convincingly show that dorsal oligodendrocytes form more sheaths due to increased sampling relative to ventral, with similar rates of sheath retraction. Finally, the authors conclude that Rab5 and Rab11 promote myelination locally and cell-autonomously in oligodendrocytes, showing specifically that Rab5 is critical for stabilization of nascent sheaths but is dispensable for sampling. Altogether, the authors provide novel and detailed visualization of early myelin sheath development by oligodendrocytes.
Strengths:
This is one of the first studies to closely examine early oligodendrocyte behavior at high resolution and adds to a body of work showing how oligodendrocytes initiate and maintain myelin sheaths. The authors find that sampling is widespread while most immature sheaths destabilize. This is an intriguing finding, as sampling is likely an energetically intensive process, but its prevalence in wild-type animals suggests that it is an important part of development.The authors' claims are substantiated by technically challenging mosaic labeling experiments that monitored individual oligodendrocyte development over the course of days. Importantly, the authors have measured the sheath accumulation and loss phase for a single oligodendrocyte over the course of several days, and can pinpoint when individual sheaths are maintained with high resolution.
The imaging acquisition and data analyses are thorough, and the labor-intensive nature of the experiments is commendable. The authors carefully use statistics to validate their conclusions, including power analyses to determine appropriate sample size.
Limitations:
Prior studies suggested that the potential for each oligodendrocyte to produce myelin sheaths is at least partially dependent upon the diameter of axons enwrapped. While axons are labeled to demonstrate ensheathment, axonal diameter is not measured, and it is unclear whether dorsal and ventral oligodendrocytes behavior could be explained by regional or individual differences in axon caliber.Other recent studies suggest that early myelination is driven by axonal factors as much as oligodendrocyte-intrinsic factors. For instance, neuronal activity stabilizes myelination for a subset of early-born neuronal types. Because of the sparse labeling techniques and focus on oligodendrocyte behavior, it is unknown how or whether axonal subtypes and activity influence early oligodendrocyte sheath sampling and stabilization.
While the authors provide a tantalizing suggestion that early oligodendrocyte sampling primes axonal segments for myelination, it is not tested directly here. Thus, the paper does not address why, or even if, repeated sampling is important in development.
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