From Schwann Cells to Melanoma: Neural Crest Derived Cells in Development and Disease

Neural crest cells (NCCs) possess the unique potential to give rise to a great variety of cell types within vertebrates. Proper NCC development is critical for survival, thus defects in specification and differentiation result in a range of neurocristopathies, which can severely impact human health. This dissertation focuses on the development and diseases of two NCC-derived cell types: Schwann cells and melanocytes. Schwann cells are the myelinating glia of the peripheral nervous system (PNS). Myelin, a lipid-rich, multi-membrane structure, is necessary for the efficient function and health of the jawed vertebrate nervous system. The importance of myelin in human health is underscored by debilitating neuropathies like Charcot-Marie-Tooth disease. Melanocytes are pigment producing cells that provide protection from deleterious UV-induced DNA damage, and positioning of melanocytes in the skin results in the wide array of pigmentation patterns among vertebrates. Failure of proper melanocyte development can lead to skin pigmentation diseases, such as Waardenburg-Shah syndrome. Additionally, inappropriate reactivation of developmental programs in melanocytes can contribute to melanoma skin cancer. The study of the development and disease of both Schwann cells and melanocytes has been greatly facilitated by the power of the zebrafish model. Chapter 1 details the common developmental origins of Schwann cells and melanocytes from the neural crest and introduces key experimental features of zebrafish used in this dissertation. Chapter 2 documents published work that examines a peripheral myelin mutant, dock1stl145, identified from a large-scale forward genetic screen in zebrafish designed to identify novel regulators of myelination. dock1 encodes a highly conserved atypical guanine nucleotide exchange factor (GEF) that activates Rac1, a Rho GTPase that is necessary for Schwann cell radial sorting and actin dynamics. The stl145 mutant contains an early stop codon in the Rac1 binding domain of Dock1. Therefore, we hypothesized that dock1 is necessary for proper radial sorting by Schwann cells and cytoskeletal dynamics. During radial sorting, Schwann cells extend processes into an axon bundle to select an axon to myelinate, requiring elaborate changes to cell shape. Once an axon is selected, a Schwann cell then extends its membrane to wrap the axon to form the myelin sheath. How these dramatic cell shape changes occur and how the actin cytoskeleton behaves in Schwann cells is still not well understood. We therefore utilized a series of dock1 mutants to better understand a potential intracellular regulator of the actin cytoskeleton in Schwann cells. Characterization of dock1 mutants revealed delays in mature myelin gene expression by whole mount in situ hybridization. Examination of the myelin ultrastructure using transmission electron microscopy found a reduction in the number of sorted and myelinated axons, indicative of radial sorting delays. We also performed the first in vivo live-imaging of the F-actin cytoskeleton in migrating Schwann cells, which did not reveal migration defects in mutant Schwann cells, suggesting that dock1 functions at the transition between when a Schwann cell stops migrating and begins radial sorting. These data inform our limited understanding of the role of GEFs during Schwann cell development and myelination. Chapter 3 investigates epigenetic regulators of melanocyte development and cancer of melanocytes, termed melanoma. Melanoma, a deadly form of skin cancer, continues to rise in incidence. Therefore, defining initiating steps of melanoma is of utmost significance. A critical step in melanoma initiation is the expression of a neural crest program, including upregulation of the neural crest transcription factor, Sox10. We hypothesize that epigenetic regulation of sox10 may be critical for re-establishing neural crest identity in melanoma initiation because of its vital role in NCC development and melanoma initiation and maintenance. Thus, using ATAC-seq analysis to profile open regions of chromatin in zebrafish melanoma tumors, we identified regulatory regions near the sox10 locus in zebrafish for a role in melanoma initiation. Activity of these sox10 putative enhancers in NCCs and melanoma was tested in a melanoma zebrafish model. Generation of stable transgenic lines for top candidate sox10 regulatory elements enabled further characterization of embryonic neural crest expression patterns and activity of these putative enhancer elements in melanoma. One element (termed peak5) derived from a region ~22.8 kb upstream of the sox10 transcriptional start site drives EGFP reporter expression in a subset of embryonic neural crest and Kolmer-Agduhr neurons, as well as specifically in early melanoma precursor lesions and tumors. Within peak5, we identified a region conserved across members of the Cyprinidae family (a family of freshwater fish) that is required for peak5 neural crest activity, and hypothesize it may function as a critical enhancer in driving sox10 expression during melanomagenesis. Within this conserved region, dimeric SoxE binding sites are necessary for peak5 activity. To test the endogenous function of peak5 in melanoma initiation, we generated several CRISPR-mediated deletion alleles, which we continue to analyze. Together, these data enhance our understanding of epigenetic mechanisms that drive the re-emergence of neural crest identity in melanoma. Collectively, this dissertation harnesses the power of the zebrafish model to deepen our understanding of Schwann cell development and melanocyte development and disease. While Schwann cells and melanocytes are derived from a common precursor, the studies within this dissertation highlight how the unique traits and functions of neural crest derived cells are influenced by both intracellular proteins and cis¬-regulators in the genome. Understanding how coding and non-coding regions of the genome guide NCC development and contribute to the malfunction of neural crest derived cells in disease can provide a basic foundation to inform treatment of human neurocristopathies