Ovarian germline stem cell dedifferentiation is cytoneme dependent

Progenitor cell dedifferentiation is important for stem cell maintenance during tissue repair and age-related stem cell decline. Here, we use the <i>Drosophila</i> ovary as a model to study the role of cytonemes in bone morphogenic protein (BMP) signaling-directed germline stem cell (GSC) maintenance and dedifferentiation of germ cells to GSCs. We provide evidence that differentiating germ cell cysts extend longer cytonemes that are more polarized toward the niche during dedifferentiation to reactivate BMP signaling. The presence of additional somatic cells in the niche is associated with a failure of germ cell dedifferentiation, consistent with the formation of a physical barrier to cytoneme-niche contact and outcompetition of germ cells for BMP. Using BMP beads in vitro, we show that these are sufficient to induce cytoneme-dependent contacts in <i>Drosophila</i> tissue culture cells. We demonstrate that the Enabled (Ena) actin polymerase is localized to the tips of germ cell cytonemes and is necessary for robust cytoneme formation, as its mislocalization reduces the frequency, length, and directionality of cytonemes. During homeostasis, specifically perturbing cytoneme function through Ena mislocalization impairs GSC fitness by reducing GSC BMP signaling and niche occupancy. Disrupting cytonemes by targeting Ena during dedifferentiation reduces germ cell BMP responsiveness and the ability of differentiating cysts to dedifferentiate. Overall, our results provide evidence that cytonemes play a fundamental role in establishing polarized signaling and niche occupancy during stem cell maintenance and dedifferentiation.