The ability to generate complete genomic sequences will provide unique opportunities to explore how variation impacts evolution and human health, to discover mechanisms regulating organism development, and to manage disease diagnosis and intervention. The effective utilization of this genomic information requires a detailed understanding of the function of encoded information. While tremendous progress has been made in defining individual components of genomic sequences, we still do not understand the function of most annotated genes, we have a limited understanding of the role of non-coding sequences in gene regulation, and we have just started to define the contribution of genomic alterations to human disease. Our section directly addresses these issues by using genomic tools and genetic manipulation of model organisms to unravel genome function and to dissect gene regulatory pathways in development and disease. We integrate data from basic science with clinical information to: 1) identify pathways that regulate mammalian development, 2) understand how alterations in these pathways lead to disease states, and 3) develop paradigms for therapeutic interventions. Our group has demonstrated that mice heterozygous for mutations in the transcription factor SOX10 exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin and absence of myenteric ganglia in the colon. We have also shown that SOX10 homozygous mutants die in utero and also exhibit extensive defects in the entire peripheral nervous system. The human congenital disorder Hirschsprung disease can be caused by SOX10 mutations, and it also exhibits rectocolic aganglionosis and can be associated with hypopigmentation. Thus SOX10 mice serve as mouse models for human disease as well as broadly inform us about neural crest development and disease. Our goal is to understand the function of SOX10 in mammalian development and use this information to understand the pathology of and develop treatments for neural crest disorders. 1. SOX10 function in vivo. During embryogenesis, the transcription factor Sox10 drives the survival and differentiation of the melanocyte lineage. However, the role that SOX10 plays in postnatal melanocytes is not established. We have now shown in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Loss of Sox10 expression results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10) mice) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and SOX10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of SOX10 hair graying is through increased expression of MITF, a target of SOX10, by asking if haploinsufficiency for Mitf can rescue hair graying in Tg(DctSox10) animals. Surprisingly, the presence of a mutant allele of Mitf (Mitfvga9) does not mitigate but exacerbates Tg(DctSox10) hair graying, suggesting that MITF participates in the negative regulation of Sox10 in McSCs. This demonstrated that while SOX10 is necessary to maintain the postnatal melanocyte lineage, it is simultaneously prevented from driving differentiation in the McSCs. These data illustrated how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage. This supports our hypothesis that alterations in SOX10 function can be used to alter neural crest cell function, and furthermore may provide a paradigm for intervention in melanoma. 2. SOX10 modifier pathways. We have established a whole genome mutagenesis program to identify genetic factors that interact with SOX10. We have identified over 8 functional mutations that affect varying aspects of melanocyte development. Included in these was a mutation in the mouse gene encoding ribosomal protein S7 (Rps7) in mouse, a gene implicated in Diamond Blackfan Anemia (DBA) in humans. Ribosomes are composed of two subunits that each consist of a large number of proteins, and their function of translating mRNA into protein is essential for cell viability. Naturally occurring or genetically engineered mutations within an individual ribosomal protein provide a valuable resource, since the resulting abnormal phenotypes reveal the function of each ribosomal protein. A number of mutations recently identified in mammalian ribosomal subunit genes have confirmed that homozygous loss of function consistently results in lethality;however, haploinsufficiency causes a variety of tissue-specific phenotypes. We showed that Rps7 haploinsufficiency causes decreased size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations, phenotypes that also occur with haploinsufficiency for other ribosomal subunits. Additionally, significant apoptosis occurs within the developing central nervous system (CNS) along with subtle behavioral phenotypes, suggesting RPS7 is required for CNS development. Although mutation of mouse Rps7 did not present an analogous phenotype to that of human DBA, the phenotypes we observed in the Rps7 mouse mutants indicate RPS7 should be considered as a candidate for a broader spectrum of human diseases. We also examined Magoh, another gene from our SOX10 interaction screen that we discovered to be important in melanoblast development. Melanoblasts are a population of neural crest-derived cells that generate the pigment-producing melanocytes of our body. Defective melanoblast development and function underlies many disorders, including Waardenburg syndrome and melanoma. Understanding the genetic regulation of melanoblast development will help elucidate the etiology of these and other neurocristopathies. We demonstrated that Magoh, a component of the exon junction complex, is required for normal melanoblast development. Magoh haploinsufficient mice were hypopigmented and exhibited robust genetic interactions with SOX10. These phenotypes were caused by a marked reduction in melanoblast number beginning at mid-embryogenesis. Strikingly, while Magoh haploinsufficiency severely reduced epidermal melanoblasts, it did not significantly affect the number of dermal melanoblasts. These data indicated Magoh impacts melanoblast development by disproportionately affecting expansion of epidermal melanoblast populations. We probed the cellular basis for melanoblast reduction and discovered that Magoh mutant melanoblasts did not undergo increased apoptosis, but instead were arrested in mitosis. Mitotic arrest was evident in both Magoh haploinsufficient embryos and in Magoh siRNA-treated melanoma cell lines. Together our findings indicated that Magoh-regulated proliferation of melanoblasts in the dermis may be critical for production of epidermally-bound melanoblasts. Our results point to a central role for Magoh in melanocyte development.

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Hwang, Hun-Way; Baxter, Laura L; Loftus, Stacie K et al. (2014) Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A. Pigment Cell Melanoma Res 27:777-87
Watkins-Chow, Dawn E; Cooke, Joanna; Pidsley, Ruth et al. (2013) Mutation of the diamond-blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes. PLoS Genet 9:e1003094
Rusmini, Marta; Griseri, Paola; Lantieri, Francesca et al. (2013) Induction of RET dependent and independent pro-inflammatory programs in human peripheral blood mononuclear cells from Hirschsprung patients. PLoS One 8:e59066
Cronin, Julia C; Wunderlich, John; Loftus, Stacie K et al. (2009) Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res 22:435-44
Silver, Debra L; Hou, Ling; Somerville, Robert et al. (2008) The secreted metalloprotease ADAMTS20 is required for melanoblast survival. PLoS Genet 4:e1000003