PROJECT SUMMARY / ABSTRACT Integration of the jaw with the surrounding musculature is essential for speech and mastication. A fundamental step in jaw integration begins in development, with formation of stable tendon-bone attachments that are zonally organized into tendon, fibrocartilage, mineralized fibrocartilage, and bone. The gradient of skeletogenic cell types within the attachment arises from attachment progenitors (APs) that, through unclear mechanisms, interpret chondrogenic versus tenogenic signaling to acquire distinct cell fates along the tendon- bone axis. Even less is known about APs of the jaw which, unlike their counterparts in the limb and trunk, are derived from neural crest cells (NCC). This study tests the idea that jaw APs differentiate into a gradient of skeletogenic cell types through a series of binary switches that are regulated by an NCC-specific mechanism. We have found that jaw APs express graded levels of Scx, Runx2, and Sox9 depending on their position along the tendon-bone axis. We also found that during AP differentiation a novel intermediate Scx+/Runx2+ population emerges. In Runx2+/- mice this intermediate population fails to form, and APs differentiate into tendon over cartilage/bone. While this suggests that tripotent APs differentiate through lineage-restricted intermediates, how APs spatially interpret signals for tendon vs. cartilage/bone to make these cell fate decisions and whether APs always choose between one fate or the other (e.g. tenocyte vs. osteoblast) or acquire hybrid properties (e.g. osteofibrogenic) is unknown. We recently showed that an Fgf-Notch signaling axis is regionally deployed along the tendon-bone interface and promotes AP differentiation into tendon over cartilage/bone. This mechanism appears NCC-specific, as loss of Fgfr2 in mesoderm-derived APs does not alter limb attachment development. In this study, we use mouse genetics along with cutting-edge genomics to test that, during AP differentiation, integration of Fgf and Notch signaling promotes tendon cell fate in a series of binary switches by regulating levels of Scx, Runx2, and Sox9 transcription. In Aim1 we will use clonal lineage tracing and scRNA-seq to determine the lineage relationship between APs and the skeletogenic cells in the tendon-bone attachment. In Aim2, we will use conditional mouse genetics to determine how differences in Notch signal strength along the tendon-bone axis alter AP cell fate decisions. In Aim3, we will use a combination of mouse genetics and CUT&RUN-seq to test that Erk signaling integrates Fgf and Notch signaling through linear and parallel mechanisms. In the linear mechanism, Erk activates Notch2 signaling by initiating Dll1 expression. In the parallel mechanism, Erk and Notch2 independently activate the same downstream targets genes for tendon fate including Scx. Completion of these aims will reveal a developmental mechanism that establishes a gradient of skeletogenic cell types in tendon-bone attach...