Master of Science
Dr. Gregory Kelly
Mouse F9 cells differentiate into primitive endoderm (PrE) when treated with retinoic acid (RA) and this is accompanied by the upregulation of Wnt6 and activation of the canonical WNT/β-catenin pathway. Previous studies have demonstrated the necessity of β-catenin-TCF/LEF transcription for primitive endoderm differentiation, however the Frizzled (FZD) receptor responsible for binding WNT6 and activating the canonical WNT pathway is not known. It was hypothesized that FZD7 is responsible for binding and transducing the WNT6 signal. Fzd7 mRNA was detected in undifferentiated and primitive endoderm cells, and its expression does not change significantly in response to RA. Moreover and contrary to my hypothesis, the knockdown of endogenous Fzd7 with siRNA does not attenuate differentiation. Other Fzd receptors are expressed in F9 cells and notably Fzd1, 4 and 8 are significantly upregulated by RA, and it is possible that one or more of these are serving to induce the WNT6 signal. That said, this study supports the notion that canonical WNT signaling activated through FZD receptors is necessary for PrE differentiation as the inhibitor Dickkopf-1 (Dkk1) is upregulated in response to RA, and expressing human DKK1 in undifferentiated F9 cells or treating cells with DKK1 conditioned media impedes the ability of RA to induce PrE. Together, this data indicates that FZD receptors are involved in WNT6 signal transduction in F9 cells and attenuating canonical WNT signaling using DKK1 blocks the ability of RA to induce PrE.
Golenia, Gregory M., "Wnt Signalling During F9 Cell Differentiation" (2015). Electronic Thesis and Dissertation Repository. 3249.