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. Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC. 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. One of these mutations causes neural tube clsure defects and was investigated in a Dev Neurbiol paper. Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155(msp3) , a unique ENU-induced allele in mice. Homozygous Baf155(mps3) embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. 3. SOX10 and adult stem cell genetics. Hair graying in mouse is attributed to the loss of melanocyte stem cell function and the progressive depletion of the follicular melanocyte population. Single-gene, hair graying mouse models have pointed to a number of critical pathways involved in melanocyte stem cell biology; however, the broad range of phenotypic variation observed in human hair graying suggests that additional genetic variants involved in this process may yet be discovered. Using a sensitized approach, we ask here whether natural genetic variation influences a predominant cellular mechanism of hair graying in mouse, melanocyte stem cell differentiation. We developed an innovative method to quantify melanocyte stem cell differentiation by measuring ectopically pigmented melanocyte stem cells in response to the melanocyte-specific transgene Tg(Dct-Sox10). We make the novel observation that the production of ectopically pigmented melanocyte stem cells varies considerably across strains. The success of sensitizing for melanocyte stem cell differentiation by Tg(Dct-Sox10) sets the stage for future investigations into the genetic basis of strain-specific contributions to melanocyte stem cell biology.
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