Glial-dependent clustering of voltage-gated ion channels in Drosophila precedes myelin formation
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eLife assessment
The authors set out to characterize a function of Drosophila glia that form an expansion of myelin-like membranes that might facilitate rapid nerve conduction. A combination of Drosophila genetics, antibody staining, and electron microscopy is used to characterize this "myelin" and the role of glial wrapping in clustering of sodium/potassium channels at motor exit points. The results are valuable, as they would point to Drosophila as a new, genetically accessible model organism to study myelin evolution. While the results are interesting, the strength of the evidence provided is incomplete due to inadequate quantification of the data provided.
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Abstract
Neuronal information conductance often involves the transmission of action potentials. The spreading of action potentials along the axonal process of a neuron is based on three physical parameters: the axial resistance of the axon, the axonal insulation by glial membranes, and the positioning of voltage-gated ion channels. In vertebrates, myelin and channel clustering allow fast saltatory conductance. Here, we show that in Drosophila melanogaster voltage-gated sodium and potassium channels, Para and Shal, co-localize and cluster in an area resembling the axon initial segment. The local enrichment of Para but not of Shal localization depends on the presence of peripheral wrapping glial cells. In larvae, relatively low levels of Para channels are needed to allow proper signal transduction and nerves are simply wrapped by glial cells. In adults, the concentration of Para increases and is prominently found at the axon initial segment of motor neurons. Concomitantly, these axon domains are covered by a mesh of glial processes forming a lacunar structure that possibly serves as an ion reservoir. Directly flanking this domain glial processes forming the lacunar area appear to collapse and closely apposed stacks of glial cell processes can be detected, resembling a myelin-like insulation. Thus, Drosophila development may reflect the evolution of myelin which forms in response to increased levels of clustered voltage-gated ion channels.
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Author Response
Reviewer #2 (Public Review):
In Rey et al., the authors goal was to characterize the development of a myelin-like (lacunar) expansion of glial membrane in Drosophila. Although myelin is largely considered a vertebrate innovation, there are a handful of invertebrate models that have been described with glial-derived "myelin," though these systems are not amenable to the same genetic control as Drosophila. To that end, the authors first newly-developed genetics and antibodies to characterize the presence of an axon initial segment (AIS) for adult Drosophila motor neurons that is present at the border between the central and peripheral nervous systems. They show that both sodium (Para) and potassium (Shal) channels, which are typically enriched at the AIS in mammalian neurons, are enriched at this border specifically on …
Author Response
Reviewer #2 (Public Review):
In Rey et al., the authors goal was to characterize the development of a myelin-like (lacunar) expansion of glial membrane in Drosophila. Although myelin is largely considered a vertebrate innovation, there are a handful of invertebrate models that have been described with glial-derived "myelin," though these systems are not amenable to the same genetic control as Drosophila. To that end, the authors first newly-developed genetics and antibodies to characterize the presence of an axon initial segment (AIS) for adult Drosophila motor neurons that is present at the border between the central and peripheral nervous systems. They show that both sodium (Para) and potassium (Shal) channels, which are typically enriched at the AIS in mammalian neurons, are enriched at this border specifically on motor neurons. They then used multiple types of transmission electron microscopy to visualize this region and found that along with clustering of channels, there is an expansion of membranes from wrapping glia that is reminiscent of myelin. At times, this expansion spirally wraps around larger axons. Finally, they show that genetic ablation of wrapping glia results in an upregulation and redistribution of Para.
Major strengths of this manuscript include the creation of new genetic tools for visualization of subcellular features (e.g. channels) by both light microscopy and electron microscopy.
While this manuscript provides an interesting set of data, but suffers from a lack of quantification and annotation to allow the reader to judge whether this is a robust phenomenon. To increase the reader's confidence in these studies, substantially more quantification of the data is required.
Furthermore, to improve the accessibility of this manuscript, I have the following suggestions:
- Please label the panels throughout the figures with an abbreviated genotype and what the fluorophores signify. Similarly, the presence of scale bars in uneven across the figures.
This was all corrected.
- For panels where only one channel is shown, please show these in black and white, which is easier for the visually-impaired.
We have not done this, since the color adds another layer of information (e.g. paramCherry is in magenta, whereas anti-Para staining is in green) which in our view helps to make the complex figures easier to understand.
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eLife assessment
The authors set out to characterize a function of Drosophila glia that form an expansion of myelin-like membranes that might facilitate rapid nerve conduction. A combination of Drosophila genetics, antibody staining, and electron microscopy is used to characterize this "myelin" and the role of glial wrapping in clustering of sodium/potassium channels at motor exit points. The results are valuable, as they would point to Drosophila as a new, genetically accessible model organism to study myelin evolution. While the results are interesting, the strength of the evidence provided is incomplete due to inadequate quantification of the data provided.
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Reviewer #1 (Public Review):
This study characterizes the localization of the lone voltage-gated Na channel in Drosophila Para in motor and sensory neurons. Like previous studies, the authors identify an enrichment of Na (and importantly the K+ channel Shal) in axon initial segment-like (AIS-) areas in motor neuron axons, and show that this structure is not apparent in axons of sensory neurons. Upon ablation of wrapping glia in the periphery, the authors find this AIS-like organization of Para is lost. Finally, compelling EM analyses of peripheral nerves suggest an intriguing area devoid of glia around AIS-like structures, and some evidence for myelin-like structures along the distal axon. The author propose several interesting ideas for how these structures might be involved in AP signaling and as evolutionary precursors to …
Reviewer #1 (Public Review):
This study characterizes the localization of the lone voltage-gated Na channel in Drosophila Para in motor and sensory neurons. Like previous studies, the authors identify an enrichment of Na (and importantly the K+ channel Shal) in axon initial segment-like (AIS-) areas in motor neuron axons, and show that this structure is not apparent in axons of sensory neurons. Upon ablation of wrapping glia in the periphery, the authors find this AIS-like organization of Para is lost. Finally, compelling EM analyses of peripheral nerves suggest an intriguing area devoid of glia around AIS-like structures, and some evidence for myelin-like structures along the distal axon. The author propose several interesting ideas for how these structures might be involved in AP signaling and as evolutionary precursors to conventional myelination and saltatory conductance in vertebrates.
Clearly, the evolution of myelination, and how glia contribute to neuronal firing in systems without classically accepted myelination and saltatory conductance are important questions. Although much of the Para clustering in AIS-like domains and regular densities along motor axons have been described in previous studies, the ultrastructural analyses and dependence on wrapping glia might be important advances in the field. In particular, major strengths of this study are the detailed analysis of AIS-like Para clusters, spanning molecular genetic, confocal and super resolution imaging, and ultrastructural approaches and clear writing. However, these strengths are somewhat tempered by a lack of functional approaches to test the idea of a lacunar structure that promotes ion exchange at putative AIS regions as well as little mechanistic insight into how glia may specifically coordinate the formation of Para clusters in AIS-like regions.
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Reviewer #2 (Public Review):
In Rey et al., the authors goal was to characterize the development of a myelin-like (lacunar) expansion of glial membrane in Drosophila. Although myelin is largely considered a vertebrate innovation, there are a handful of invertebrate models that have been described with glial-derived "myelin," though these systems are not amenable to the same genetic control as Drosophila. To that end, the authors first newly-developed genetics and antibodies to characterize the presence of an axon initial segment (AIS) for adult Drosophila motor neurons that is present at the border between the central and peripheral nervous systems. They show that both sodium (Para) and potassium (Shal) channels, which are typically enriched at the AIS in mammalian neurons, are enriched at this border specifically on motor neurons. They …
Reviewer #2 (Public Review):
In Rey et al., the authors goal was to characterize the development of a myelin-like (lacunar) expansion of glial membrane in Drosophila. Although myelin is largely considered a vertebrate innovation, there are a handful of invertebrate models that have been described with glial-derived "myelin," though these systems are not amenable to the same genetic control as Drosophila. To that end, the authors first newly-developed genetics and antibodies to characterize the presence of an axon initial segment (AIS) for adult Drosophila motor neurons that is present at the border between the central and peripheral nervous systems. They show that both sodium (Para) and potassium (Shal) channels, which are typically enriched at the AIS in mammalian neurons, are enriched at this border specifically on motor neurons. They then used multiple types of transmission electron microscopy to visualize this region and found that along with clustering of channels, there is an expansion of membranes from wrapping glia that is reminiscent of myelin. At times, this expansion spirally wraps around larger axons. Finally, they show that genetic ablation of wrapping glia results in an upregulation and redistribution of Para.
Major strengths of this manuscript include the creation of new genetic tools for visualization of subcellular features (e.g. channels) by both light microscopy and electron microscopy.
While this manuscript provides an interesting set of data, but suffers from a lack of quantification and annotation to allow the reader to judge whether this is a robust phenomenon. To increase the reader's confidence in these studies, substantially more quantification of the data is required.
Furthermore, to improve the accessibility of this manuscript, I have the following suggestions:
1. Please label the panels throughout the figures with an abbreviated genotype and what the fluorophores signify. Similarly, the presence of scale bars in uneven across the figures.
2. For panels where only one channel is shown, please show these in black and white, which is easier for the visually-impaired.
Overall, the description of "myelin" in Drosophila would open up the field of myelin biology to a new model system to study the molecular mechanisms that facilitated the evolution of this important glial structure. Thus, further analysis of the data would be advantageous.
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