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Date of Award
Doctor of Philosophy (PhD)
Myelin is a multilamellar sheath made by specialized glial cells that iteratively spiral and compact their plasma membranes around axon segments. In vertebrate nervous systems, myelination facilitates rapid action propagation and provides trophic support critical for neuronal survival. In the central nervous system (CNS), oligodendrocytes (OLs) extend many processes to simultaneously ensheath multiple axons, while in the peripheral nervous system (PNS), myelinating Schwann cells (SCs) pair 1:1 with a single axon segment. Elaboration of the myelin sheath is one of the most exquisite and complex examples of massive coordinated cellular shape changes in the vertebrate nervous system. Furthermore, the importance of myelinating glia is highlighted by the debilitating symptoms resulting from disruptions of myelination in diseases including multiple sclerosis, and peripheral neuropathies such as Charcot-Marie-Tooth disease. And yet, when compared to their neuronal counterparts, relatively little is known about the molecular machinery governing the development of myelinating glia. In my thesis work, I used a combination of classic genetics and the latest genomics approaches in both zebrafish and mouse models to identify novel regulators of myelinating glial cell development. Recent work from the Monk lab, and others, has shown that the adhesion family of G protein-coupled receptors (aGPCRs), particularly Gpr126 and Gpr56, mediate cell-cell and cell-matrix interactions to regulate SC and OL development. Importantly, the roles of both Gpr126 and Gpr56 in myelinating glia were first discovered in zebrafish, and the zebrafish has proven to be a useful model for studying several other aGPCRs in various contexts. However, despite this, the full repertoire of aGPCRs and their expression profiles in zebrafish were unknown, limiting the utility of the zebrafish model for the aGPCR field. Therefore, I mined the zebrafish genome to define the aGPCR repertoire in zebrafish and used high throughput qPCR to characterize the expression profiles of all aGPCRs throughout development and in adult tissues. While humans have 33 known aGPCRs, and mice and rats have 31 each, we identified at least 59 aGPCRs in zebrafish, representing homologs of 24 of the 33 human aGPCRs. Interestingly, we found that the expression profiles of zebrafish aGPCRs closely match those of their mammalian orthologs in adult tissues, and our study was the first to describe aGPCR expression during development in any species. I also asked if any other aGPCRs might be important in myelinating glia, therefore I profiled the expression of all zebrafish aGPCRs in the optic nerve, which should enriched be for OLs, and the posterior lateral line nerve (pLLn), which is enriched for SCs. Excitingly, we showed that several other aGPCRs are highly enriched in myelinating glia-rich tissues, and future work will determine if these are also important players in myelinating glial cell development. In a separate, but complementary line of work, I collaborated with members of the Monk and Solnica-Krezel laboratories to conduct a large-scale forward genetic screen in zebrafish to identify novel genetic regulators of SC and OL development. In total we screened nearly 700 genomes and uncovered 28 mutations affecting the distribution and/or expression levels of myelin basic protein (mbp), an essential component of the myelin sheath. One of the most striking mutants, designated stl64, displayed dramatic overexpression of mbp. Using an in-house analysis pipeline, I determined that the stl64 lesion introduces a premature termination codon in the gene fbxw7. Fbxw7 is an E3 ubiquitin ligase normally responsible for maintaining proper protein homeostasis by recognizing target proteins and catalyzing their ubiquitination and subsequent degradation by the proteasome. Previous work from the Appel lab showed that Fbxw7 regulates Notch and mTOR to control OL development and myelination, but a role for Fbxw7 in SCs has never been described. Using zebrafish and SC-specific knockout models in mice, I found that Fbxw7 is critical at nearly every stage of SC development and myelination. Loss of Fbxw7 in SCs results in enhanced SC number, segregation of axons, myelin thickness, and basal lamina production. We showed that the enhancements in SC numbers, radial sorting, and myelin thickness are all due to elevated levels of mTOR signaling. When mTOR is deleted from mutant SCs, these phenotypes are rescued. Additionally, I discovered that Fbxw7 also limits the myelination capacity of SCs. Mutant SCs are capable of generating myelin around multiple axons simultaneously, in addition to being able to myelinate large axons while encompassing many small unmyelinated axons at the same time. This is the first ever in vivo description of multi-axonal myelination by SCs. Very excitingly, we found that myelination capacity of SCs is independent of mTOR, as loss of mTOR in Fbxw7 mutant SCs does not restore proper 1:1 SC-axon ratios. This suggests that a different Fbxw7 target is responsible for limiting SC myelination capacity, and ongoing work is focused on discovering the identity of that target as well as understanding the consequences and potential utility of multi-axonal myelination by Scs. In sum, I have used a combination of classic genetics as well as the latest genomics techniques to generate several tools for the aGPCR, zebrafish, and myelin research communities; and most recently I have defined a novel critical regulator of SC development and myelination, Fbxw7.
Chair and Committee
Kelly R. Monk
Valeria Cavalli, Aaron DiAntonio, David H. Gutmann, Stephen L. Johnson,
Harty, Breanne Leigh, "Genetic and genomic dissections of myelinating glial cell development" (2017). Arts & Sciences Electronic Theses and Dissertations. 1109.
Available for download on Monday, April 22, 2019