PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) signaling is essential for the development of many tissues and organs. Hh ligands are morphogens and thus direct cell fate decisions in a manner dependent on signaling strength. Precise regulation of this signaling strength is critical for embryonic development, as even modest disruptions in the signaling amplitude can result in severe birth defects. Many studies have shown that signaling strength is influenced by both morphogen concentration and duration of morphogen exposure. However, an equally important layer of regulation remains unrecognized and understudied: How does a target cell regulate its sensitivity to a morphogen? The central focus of my postdoctoral research has been to study the mechanisms that modulate a cell’s sensitivity to extracellular morphogens. Initially, I used genome-wide CRISPR screens to discover three novel Hh signaling attenuators: Mosmo, Megf8, and Mgrn1. Through support from the K99, I showed that these three proteins form a membrane-tethered ubiquitin ligase complex (the MMM complex) that suppresses a cell’s sensitivity to Hh morphogens by clearing the Hh effector Smoothened (SMO) from the cell surface and primary cilium. Using knockout mouse models, I also showed that the MMM complex is essential for proper left-right patterning and embryonic heart development. Collectively, my work has begun to (1) unravel a new mechanism through which target cells modify their responses to extracellular cues by altering the landscape of proteins at the cell surface and (2) shed light on the mechanisms that underlie the complex genetics of heterotaxy and congenital heart defects. COVID-19 related research restrictions and university closures severely delayed my career plans and personal development. While my research progress reflects the successful completion of Aims 2 and 3 in my original proposal, COVID-19 research restrictions severely delayed progress on Aim1, which sought to determine the mechanism through which the MMM complex regulates protein trafficking events. A funding extension would allow me to develop critical new skills in mass spectrometry and advanced microscopy to understand how the MMM complex regulates the trafficking of proteins to the cell surface and primary cilium. I will accomplish this with training from my mentor Dr. Rajat Rohatgi (biochemistry), my co-mentor Dr. Tim Stearns (cilia biology), and Dr. Ryan Leib (the proteomics director of the Stanford mass spectrometry core facility and member of my scientific advisory committee). A funding extension would also allow me to further train under Dr. Cecilia Lo, to become more proficient at analyzing embryonic mouse hearts for developmental defects. In summary, the training and mentorship I will receive during the extended K99 period will equip me with the knowledge necessary to transition into the study of the molecular mechanisms that contribute to heterotaxy and congenital heart defects. Ultimately, this training wi...