Cellular and genetic analysis of central nervous system myelination in zebrafish

The myelin sheath is a plasma membrane extension of specialized glial cells that wraps around neuronal processes, called axons: in so doing, myelin permits the rapid conduction of nerve impulses. Damage to myelin causes the symptoms of many human diseases including multiple sclerosis (MS) and Charcot-Marie-Tooth (CMT) neuropathies. Myelin formation (myelination) is a much more efficient mechanism than the alternative way to increase nerve conduction, namely increasing axon diameter. Large diameter axons take up space, which constrains the size and complexity of organism that can evolve using only this strategy. It is fair to say, therefore, that complex nervous systems, such as our own, have evolved in large part due to the properties of the myelin sheath. Understanding the mechanisms that control myelination is thus of both fundamental biological and medical relevance. The zebrafish is a powerful model organism in which to dissect the cellular and genetic basis of myelination. Zebrafish embryos are transparent, and tools exist to watch fluorescently labeled cells behave in real time in the living organism, at a level of detail that is not feasible in other vertebrate laboratory animals. A second major attraction of the zebrafish is the ability to carry out large-scale affordable genetic screens to find genes required for specific biological processes. In a genetic screen carried out in our lab we identified 10 genes required for the development of myelinated axons. Although we have learned a great deal our screen certainly did not have the scope to identify all the genes that regulate myelination. Our current understanding of the genetic and cellular basis of myelin formation in the central nervous system (the brain and spinal cord) remains particularly rudimentary. The overall goal of my proposal, therefore, is to determine the cellular and genetic basis of myelin formation in the zebrafish central nervous system. 1. I will directly observe the precise cellular interactions between axons and glial cells that culminate in myelination, by high-resolution time-lapse microscopy in zebrafish. 2. Previous studies have led to the intriguing hypothesis that the level of neuronal activity can regulate myelin production, which may represent a fundamental mechanism by which localized brain activity could enhance nervous system function. I will test this hypothesis in intact animals for the first time by altering levels of neural activity in zebrafish embryos and looking at the effects of different treatments on myelin production and on neurophysiology. 3. Recently a particular genetic pathway (the neuregulin-erbb pathway) has been implicated as a key regulator of myelin formation in the peripheral nervous system (the part of the nervous system outside of the brain and spinal cord), but its role in the CNS is somewhat controversial. I have exciting preliminary data that I will now fully explore that this fundamental regulatory pathway does indeed regulate myelination in the CNS. 4. We still do not know the identity of many of the genes that are required for myelination in the CNS. I will perform a new genetic screen in zebrafish and focus in particular on genes that are required for myelination in the CNS. By comparing animals with mutations in specific genes with normal animals by high-resolution analyses such as time-lapse microscopy I will be able to define exactly which aspects of myelination those genes are normally required for. I hope to set up my own independent research group at the University of Edinburgh, in laboratories that are part of a new £600m research development at the Little France Biomedical Sciences Centre. This environment will provide a world-class infrastructure, and I will be adjacent to two of the leading researchers in the field of myelin biology, which will provide an ideal environment of intellectual support and potential collaboration, to continue to unravel the mysteries of myelination.

The myelin sheath is a plasma membrane extension of oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS) that wraps around axons to allow the rapid transmission of nerve impulses. Although we know much about their early development we know much less about how these cells interact with axons at later stages to regulate myelination, especially in the CNS. This proposal will address four primary questions. 1. What are the cellular bases of myelination in the CNS in vivo? A single OL typically extends many individual processes to generate multiple myelin segments, but how this process occurs in vivo is almost entirely unclear. I will carry out time-lapse imaging to characterise the precise cell behaviors that surround myelination. I will also test if axon diameter is an intrinsic property of neurons that prefigures myelination or is regulated by OLs. 2. What is the role of neural activity during myelination in vivo? Previous studies have suggested that neuronal activity may regulate myelination, but how this occurs in vivo remains unclear. I will examine myelination following inhibition of all neural activity in vivo and create chimeric animals with both active and inactive neurons within a nerve to determine if different levels of activity can regulate myelin production. 3. How does ErbB signaling regulate myelination in the CNS? In the PNS ErbB receptor signaling is required for myelination but its role during myelination in the CNS remains unclear. We have exciting preliminary data that treatment with a pan ErbB inhibitor reduces myelination in the CNS. I will determine which ErbB receptor(s) mediate this and which aspects of CNS development and myelination require ErbB function. 4. What genes are required for myelination in the CNS? I will carry out a genetic screen in zebrafish to identify genes specifically required for late stages of OL development that surround myelination.