UC San Diego

The processes of differentiation and self renewal in human pluripotent stem cells (hPSCs) is regulated by a host of extracellular factors, many of which act through cell surface receptors and trigger signaling pathways to control gene expression. A key class of developmental signaling modules regulating stem cell behavior is the Wnt signaling pathway. Our understanding of how Wnt signaling proteins regulate the choice between self-renewal and differentiation is inadequate. Complicating these studies is the fact that the genome contains 19 WNT genes and at least 13 WNT receptors encoded by the FRIZZLED, ROR and RYK genes, which bind and transduce signaling inputs of the various Wnt proteins. My research has explored the interactions between two Wnt proteins, Wnt3a and Wnt5a, and a subset of Wnt receptor : Frizzled 5 (FZD5), Frizzled 7 (FZD7), Frizzled 10 (FZD10), and ROR2. Both FZD and ROR receptors have a conserved domain on the extracellular portion on the receptor known as the cysteine-rich domain (CRD), which is responsible for binding Wnts. I constructed fusion genes comprised of the CRD region of these Wnt receptors and a tag, the constant region of an immunoglobulin (Ig). Upon stable transduction, selection, and clonal expansion of cells carrying these chimeras, I isolated and purified the fusion proteins. Finally, I used these recombinant proteins to interfere with Wnt signaling in cell-based assays. My results to date demonstrate that these CRD-Ig fusion proteins antagonizeWnt3a signaling in a standard Wnt reporter assay. These studies provide proof -of-principle that this approach can be utilized to specifically affect Wnt signaling in cell-based assays and can potentially be exploited to regulate stem cell behavior