(27cd) Analysis of Pmad and Medea Expression in BMP Pathway in S2 Cells and Drosophila Germline Stem Cells Niche

Understanding how cell-cell signaling affects stem cell decisions in their native context is crucial for both tissue engineering and regenerative biology. However, the relationship between signaling and differentiation is not well-characterized mechanistically or quantitatively. Therefore, in this work, we have analyzed the BMP/Dpp signaling pathway, which regulates differentiation vs. self-renewal in the Drosophila melanogaster (fruit fly) female germline stem cells (GSCs).

On the anterior side of the GSC niche, the BMP morphogen Dpp activates Type I receptor Thickveins (Tkv), and Type II receptor Punt, which then phosphorylate Mothers against Dpp (Mad, the Drosophila homolog of Smad1/5/8). Phosphorylated Mad (pMad) then binds to the cofactor Medea (Smad4) and the complex translocates to the nucleus to promote GSC self-renewal. To investigate the dynamics of the BMP signaling pathway, we have tagged Mad and Medea with eGFP and mScarlet-I. To test these constructs, we first transfected them into S2 cells and measured nuclear localization of Mad and Medea before and after the addition of Dpp ligand, which will cause the migration of these proteins into the nuclei. We will then create transgenic fly lines carrying these constructs and analyze their expression in the Drosophila GSC niche. Using optogenetic receptor constructs to activate the BMP pathway through light, we will measure their dynamics and construct a predictive, mathematical model of BMP signal transduction.