The nhr-67/tailless gene of C. elegans encodes a nuclear receptor transcription factor that plays a central role in regulating cell differentiation,and ultimately organogenesis. The human version of the nhr-67 gene, Tlx, has been shown to be a key regulator of neural stem cell identity. When the Tlx gene is compromised in vertebrates, neural stem cells cannot be maintained as dividing stem cells. Therefore, an understanding of how Tlx regulates other genes is central to understanding neural stem cells, and an important first step in identifying potential candidates for therapy in neurodegenerative disease. Towards this end, the Wightman Lab is studying the regulatory pathways both upstream and downstream of nhr-67, using the differentiation of uterine cells as a model. Among the most interesting targets of nhr-67 are the cell-cell signaling receptor Notch (lin-12) and its cognate signal delta (lag-2). In humans, Notch has been linked to developmental mechanisms associated with aortic valve disease and cancer. While Notch is also known to function in vertebrate and invertebrate neural development, this study is among the first to link Tlx function to Notch regulation. This study seeks to advance understanding of Tlx-based regulatory systems by identifying both genes that are upstream of nhr-67 and those that are downstream targets of nhr-67 function. The first major aim, to determine the upstream regulators of nhr-67 transcription in the ventral uterus, will evaluate candidate upstream transcription factors (bHLH heterodimers, LAG-1 mediator of Notch response, SEX-1 nuclear receptor) through a combination of in vitro and in vivo approaches. The bHLH transcription factor, HLH-2, which has a well-defined role in early ventral uterine development, will be evaluated in vivo as a candidate direct activator of nhr-67 expression. The study will investigate how a Notch- based feedback loop sets transcriptional levels of nhr-67, and test the hypothesis that the sex-1 embryonic sex determination function is complemented by a later function in sex-specific organogenesis. The second major aim, to evaluate the downstream mediators of nhr-67 function in the ventral uterus, will identify and study potential effectors of NHR-67 for differentiation events. This aspect of the project will clarify the role of Notch regulation as a key feature of nhr-67 function, examine the relationship between nhr-67 and a globin gene, glb-12, and identify unanticipated new targets using a new nuclei purification and differential expression strategy, combined with an established cell-sorting strategy. Successful completion of the differential expression strategy will demonstrate how a method by which other researchers can study gene expression in a small number of cells. These molecular and genetic strategies will provide a better understanding of the regulatory events that govern important developmental events. The targets identified by this study will provide important candidates for future investigations of the molecular pathways that control specification of neural stem cells and organogenesis.
The nhr-67 gene of C. elegans is the equivalent to the human Tlx gene, which is required for neural stem cell identity. The study of the mechanisms of nhr-67 function will inform on key conserved pathways that are important for understanding regulation of neural stem cell development, which may help identify targets for therapies to treat neurodegenerative disease. An understanding of the regulation of the Notch gene helps an understanding of the mechanisms of human disease, since Notch has a well-established role in regulating vascular morphogenesis and cancer progression in vertebrates.