Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.

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Thesis advisor: Laura Anne Lowery / Wolf Hirschhorn Syndrome (WHS) is a neurodevelopmental disorder characterized by craniofacial malformations, heart and skeletal defects, intellectual disability as well as seizure disorders. While this disorder is thought to arise from a deletion of a region on the short arm of chromosome 4, which includes the four genes WHSC1, WHSC2, LETM1 and TACC3, the mechanism by which loss of these genes results in WHS is not understood. Given that these genes have been linked to cell migration and that affected tissues include those derived from the neural crest, we propose that WHS results from a defect in neural crest cell migration. Here, we show that WHSC1, WHSC2, TACC3 and LETM1 are all expressed along the neural tube and developing neural folds during Xenopus embryonic development. These genes are additionally enriched in the pharyngeal arches, which are migrating neural crest cells. The knockdown of these WHS-related genes leads to variable defects in craniofacial and cartilage morphology. Moreover, the loss of WHS gene expression causes defects in forebrain and midbrain development. This implicates these four genes in the WHS phenotype. Further analysis of both WHSC1 and TACC3 function show that their individual knockdown causes defective neural crest cell migration both in vivo and in vitro. This supports the notion that the WHS phenotype is a result of erroneous neural crest cell motility. Our analysis shows that the WHS related genes; WHSC1, WHSC2, LETM1 and TACC3, play a role in the WHS phenotype of craniofacial malformation, skeletal abnormality, and microcephaly. Further analysis of these genes will determine the combinatorial effects of their knockdown on neural crest cell migration during embryonic development to further elucidate the mechanism through which WHS develops. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Les cellules de le crête neurale sont une population de cellules précurseurs multipotentes qui émergent aux frontières du tube neural et de l’ectoderme non neural, migrent de manière extensive dans tout l'embryon et se différencient en une variété de types de cellules, notamment les cellules pigmentaires de la peau (mélanocytes), la glie du système nerveux périphérique (y compris les cellules de Schwann formant la myéline) et les neurones et les cellules gliales du système nerveux entérique. Chacune des étapes du développement de ces cellules est sous le contrôle de stimuli externes et de facteurs de transcription étroitement régulés formant un réseau complexe. Le rôle des modifications épigénétiques et post-transcriptionnelles a également été mis en évidence, mais leurs contributions aux troubles associés sont encore mal décrites. L'objectif de ma thèse était d'étudier le rôle de l'une des modifications post-transcriptionnelles les plus rependues : le RNA editing, dans le développement normal et pathologique de la crête neurale. Nous rapportons ici que ADAR1, enzyme responsable de la modification adénosine-inosine de l'ARN, est nécessaire au développement de trois dérivés de la crête neurale : les mélanocytes, les cellules de Schwann et le système nerveux entérique. L'invalidation conditionnelle spécifique d'Adar1 dans la crête neurale chez la souris provoque une dépigmentation généralisée et l'absence de myéline des nerfs périphériques résultant d'altérations affectant la survie des mélanocytes et de la différenciation des cellules de Schwann, respectivement. Des défauts de la glie entérique ont également été mis en évidence. Ces défauts sont tous trois précédés par l’activation d’une réponse immunitaire innée médiée par l’IFN. L’invalidation concomitante de MDA5, un senseur clé de détection d'ARNs non édités, corrige les défauts de myélinisation et de pigmentation observés chez les mutants adar1, suggérant qu'ADAR1, via son activité d’editing, protège ces dérivés de la crête neurale d'une production aberrante d’IFN délétère à leur survie ou différenciation. L’ensemble de ces résultats étendent le spectre d’action d’Adar1 au développement de la crête neurale normal et pathologique. / Neural crest cells are a population of multipotent precursor cells that emerge at the borders of the neural tube, migrate extensively throughout the embryo, and differentiate into a variety of cell types including the skin pigment cells (melanocytes), glia of the peripheral nervous system (including Schwann cells that form myelin) and the neurons and glia of the enteric nervous system. Each steps of the development of these cells is under the control of external stimuli and tightly regulated transcription factors that form a complex network. The role of epigenetic and post-transcriptional modifications was also highlighted, but their contributions to related disorders are still poorly described. The aim of my PhD project was to investigate the role of one of the most common post-transcriptional modification: the Adenosine to Inosine (A to I) RNA editing, in normal and pathologic NC development. Here, we report that adenosine deaminase acting on RNA (ADAR1), responsible for A to I editing of RNA, is required for regulating the development of three neural-crest derivatives: melanocytes, Schwann cells and enteric nervous system. Neural-crest specific conditional invalidation of Adar1 in mice led to global depigmentation and absence of myelin from peripheral nerves, resulting from alterations in melanocyte survival and late differentiation of Schwann cells respectively. Defects of enteric glia is also evidenced. These defects were preceded by upregulation of an innate immune inflammatory response. Simultaneous extinction of MDA5, a key sensor for the detection of unedited RNA, rescued the pigmentation and myelin defects of Adar1 mutants, suggesting that ADAR1 safeguards a subset of neural-crest derivatives from aberrant MDA5-mediated interferon production. We thus extend the landscape of ADAR1 function to the fields of neural-crest development and disease.

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