Investigating the mechanisms of a multi-state model of Wnt signaling

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PROJECT SUMMARY ABSTRACT The WNT signaling pathway regulates numerous developmental processes and plays a critical role in the maintenance of healthy tissue and cells in adults. Moreover, dysfunction in WNT signaling has been implicated in numerous developmental disorders, neurodegeneration, and tumorigenesis. Canonical WNT signaling is typically described as a ‘binary’ system, the so-called ‘two-state’ model. In the ‘off’ state, a protein destruction complex directs the continual proteolytic degradation of β-catenin. In the ‘on’ state, in the presence of a WNT ligand, this protein complex is disassembled, allowing β-catenin to accumulate and translocate into the nucleus, thereby altering gene transcription. However, this model does not fully explain how gradients of WNT signaling activity that are present during the development and patterning of many tissues lead to precise changes in transcriptional response and cell identity. In addition, this model does not adequately explain how different WNT signaling thresholds lead to the manifestation of cancer and other pathological conditions. To better understand the complex, multifaceted role of WNT signaling in human development and disease, we have engineered an in vitro human pluripotent stem cell (hPSC)-based model that mimics the same early in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube. Using this system we will test our proposed model and hypothesis that specific levels of WNT activity are translated into precise transcriptional responses and cell phenotypes through two complementary mechanisms: (i) directly through the transcriptional regulation of genes related to A/P neural tube patterning and (ii) indirectly through the actions of the transcriptional repressor SP5. In the first aim of the proposed research, we will use single cell gene expression analysis, genome-wide expression analysis (RNA-seq), and DNA binding analysis (ChIP-seq) to define the transcriptional mechanisms by which β-catenin regulates the A/P identity of hPSC-derived neural cells. In the second aim, we will utilize a series of novel knockdown and overexpression hPSC lines in conjunction with ChIP-seq analysis to investigate the role of individual TCF/LEF proteins in regulating the regional identity of hPSC-derived neural cells. Finally, in the third aim, we will use engineered knockout and knockin hPSC lines along with ChIP-seq to establish SP5 as a mediator of WNT signaling in specifying the A/P regional identity of hPSC-derived neural cells. Overall, the new insights gained from this research will not only lead to a more thorough understanding of how WNT signaling regulates early neurodevelopment but also will have significant impact on our understanding of the role of WNT signaling in disease initiation and progression.