Date of Award
Doctor of Philosophy (PhD)
Myelin is a lipid rich, multilamellar membrane that is generated by iteratively wrapping around neuronal axons segments to allow for the rapid propagation of action potentials along the length of an axon. It is produced by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). There are multiple diseases and disorders that disrupt the myelin sheath or interfere with the oligodendrocytes or Schwann cells that make it, the most common of which are multiple sclerosis in the CNS and Charcot-Marie Tooth disease Type I in the PNS. The severe cognitive, sensory and motor defects present in many of these myelinopathies highlight the importance of myelin and the importance of studying the genes responsible for its development. The complete repertoire involved in the formation of myelin in the CNS and PNS is still incomplete. To attempt to fill in some of the gaps in this list, my thesis will explore forward and reverses genetic approaches to understanding myelination.
The development of pro-myelinating Schwann cells into mature myelinating Schwann cells is difficult to study due to the transient nature of the pro-myelinating state. Taking advantage of Gpr126 mouse mutants, whose Schwann cells arrest in the pro-myelinating state, we were able to find the genes differentially expressed between the two developmental stages. Using this reverse genetics approach, one transcription factor, Myrfl, had an interesting expression profile and was chosen for further study. In order to assess the role of Myrfl in Schwann cell development, a TALEN was used to disrupt the gene in zebrafish, creating a premature stop codon in exon 4. Characterization of this mutant revealed no phenotype in the posterior lateral line nerve, pLLN, at 3 or 5 days post fertilization (dpf) but did revealed a delay of mbp expression by in situ in the anterior lateral line nerve, aLLN, at 3 dpf. Mutants no longer had this decreased mbp expression by 5 dpf. Altogether, Myrfl appears to be a transcription factor that plays a minor role in the development of Schwann cells of the aLLN exclusively.
The largest part of this thesis is dedicated to working on the ongoing large forward genetic screen for myelin mutants in the Monk lab. As the screen continued to generate new alleles, a working method to integrate whole genome sequencing with mapping efforts was needed to speed up the mapping process in lab. We generated a protocol to utilizing genomic DNA from fixed tissue for whole genome sequencing (WGS) and created a bioinformatics pipeline for WGS-based mapping of zebrafish mutants. We tested our protocol on two known zebrafish myelin mutants and the novel stl64 allele generated in our screen. Our pipeline successfully mapped the two known mutants and linked stl64 to a small portion on chromosome 1. We were then able to identified a candidate nonsense mutation in the fbxw7 gene that phenocopied a previously described fbxw7vu56 mutant. Additional experiments further confirmed that stl64 was due to a mutation in fbxw7. Successfully mapping all three mutants gave us good evidence that out pipeline worked to identify causative lesions in mutant screen alleles.
The last chapter of this thesis builds off the creation of a protocol and pipeline for the WGS of genomic DNA from fixed tissue and the bulked segregate analysis of mutant SNP ratios indicating linkage. Work on several of the alleles that came from the screen have progressed to the point where pools of mutants and pools of siblings were sent for whole genome sequencing in an attempt to begin mapping them. All alleles except for stl172 were able to be linked to a region of a specific chromosome, or in the case of stl276, chromosomes. Good candidate non-synonymous mutations were found in these linked regions for stl90, stl91, stl145 and stl287, but confirmation with second alleles and complementation crosses has yet to be done. For stl71, stl72, stl103, stl172 and stl276 a mixture of recombination mapping with individual mutants and resequencing is recommended.
Chair and Committee
Kelly R. Monk
Kelly R. Monk, Charles K. Kaufmas, Thomas J. Baranski, Joseph D. Dougherty,
Sanchez, Nicholas Enrique, "Forward and Reverse Genetic Approaches to Understanding Myelination" (2018). Arts & Sciences Electronic Theses and Dissertations. 1574.
Available for download on Sunday, May 15, 2118