Neural crest cells are a transient population of cells which differentiate into multiple derivatives. How these derivatives become specified is not well understood but Sox10 is known to be important in many of them. We are interested in defining the precise role of Sox10 in zebrafish melanophores. Current evidence suggests that the only vital function that Sox10 performs in melanophores is to induce expression of the melanocyte master regulator mitfa (Elworthy et al. 2003). We explored a model for Sox10 function in melanophores, based upon a model for Sox10’s role in mouse sympathetic neurons (Kim et al. 2003), and tested the following predictions: as well as inducing expression of mitfa, Sox10 will repress expression of genes downstream of Mitfa thus, Sox10 must be downregulated, via Mitfa, to allow melanophore differentiation. We observed derepression of melanophore marker genes in sox10t3 mutants, supporting the hypothesis that Sox10 represses these genes in wild type melanophores. We documented Sox10/sox10 downregulation in developing melanophores and generated transgenic lines to test whether this is necessary for differentiation. Unfortunately our experimental lines did not express our transgene so we were unable to test this hypothesis. However, transgenic lines, generated as controls, which express CFP in melanophores or xanthophores will be useful tools in their own right. Finally we conducted RNA injection experiments to explore regulation of melanophore genes by Sox10 and Mitfa. We found that injection of mitfa induces expression of all our melanophore markers whereas co-injection of mitfa and sox10 does not. We also found that the 7.2 kb sox10 promoter contains six Mitf binding sites and is Mitfa responsive. Our data broadly support our original model but also suggest that it does not describe the complete network. We propose a modified model for the role of Sox10 in the genetic regulatory network controlling melanophore development.

The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第24809号 / 人健博第115号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 中尾 恵, 教授 岡 昌吾, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM