PROJECT SUMMARY Biological tissues appear to “know” their intended final sizes and achieve them precisely and robustly. While, in principle, a simple negative signaling feedback should be sufficient to explain how a given stem cell lineage regulates its cellular outputs, in reality it cannot work because most tissues are physically large, with stem cells and their progeny spread out over centimeter-scale distances. How tissues overcome the microscopic decay limits of diffusible molecular signals to breach distances orders-of-magnitude in spatial scale remains elusive. This application is inspired by our serendipitous discovery that FGF and BMP mutant mice are able to grow super-long and highly imprecise hairs that can exceed the length of normal mouse hairs by 7-fold. Our lineage analyses suggest that hair stem cells continuously replenish short-lived transit-amplifying (TA) cells spatially located nearly 1 cm away from the stem cells. Interestingly, our single-cell RNA-sequencing analyses reveal previously unappreciated heterogeneity of the intermediate epithelial progenitor cells physically located between the stem and TA cells. Through an integrated mathematical and experimental approach, this application will focus on testing our new hypothesis that dynamic equilibrium between two or more intermediate cell states and their associated cell-cell communications enable feedback information propagation over large spatial scale from TA cells to stem cells to regulate the new progenitor cell production for hair size control. The first aim of the proposed research is to profile and quantify the heterogeneity of intermediate epithelial progenitors, and computationally and experimentally determine the functional link between specific intermediate progenitor states in the hair follicle and the hair length and its precision. The second aim is to define the cell-cell communication networks within the epithelial hair follicle lineage, and computationally and experimentally establish how multiple short-range signaling activities coordinate to form a long-range feedback mechanism that controls progenitor flux between distant stem and TA cell compartments for proper hair growth. The third aim is to determine the signaling impact of mesenchymal niche cells, which surround the hair follicle, on the epithelial lineage cells for hair size control. The study premise is based on novel and extensive preliminary experimental and computational data. The proposed studies are significant because they will establish new long-range signaling mechanisms and uncover novel roles of intermediate cell states in tissue size control. The proposed studies are innovative because they will establish new experimental models for studying tissue size regulation using super-long and extra-short hair mutant mice and will result in numerous new genetic mouse tools for epithelial stem cell research. They will also result in several novel mathematical and computational tools for analyz...