PROJECT SUMMARY The cranial neural crest (NC) contributes to the formation of many craniofacial structures including the bones and cartilage of the face, tooth dentin, and peripheral ganglia. Cell signaling regulates different aspects of cranial NC specification, epithelial-to-mesenchymal transition (EMT), and differentiation and disruptions in this developmental program result in many cranial NC-derived craniofacial birth defects including craniosynostosis, Treacher Collins and CHARGE syndromes, and cleft palate. BMP signaling plays a crucial role during the specification and differentiation of cranial NC, and more recently, BMP signaling was shown to control cranial NC EMT. A mechanistic understanding of the role of BMP signaling during cranial NC development is essential to develop novel preventative and therapeutic measures against craniofacial defects. Under the support of this parent award, I have made significant progress toward understanding the mechanisms of BMP signaling underlying cranial NC EMT and migration. My preliminary results suggest that BMP signaling peaks during cranial NC cells during EMT, whereas analysis of fixed sections suggest that signaling level are reduced at earlier (during specification) and later (during migration) stages (Piacentino et al., 2021). To understand the role of BMP signaling during this phase, I have inhibited BMP signaling using a dominant negative Type I BMP receptor (dnBMPR1A) and demonstrated that BMP signaling is essential for cranial NC migration, independent of specification or delamination. Furthermore, I have identified and validated novel targets of BMP signaling using a comparative transcriptome profiling approach (Piacentino et al., 2021). Unfortunately, the COVID-19 pandemic led to university closures and research restrictions that have severely delayed my career development. While I have remained productive and have published results toward the goals of the original proposal (Piacentino et al., 2021), and have identified a novel endocytic mechanism that regulates BMP signaling in cranial NC cells (Piacentino et al., 2020) [Preprint], pandemic setbacks have prevented me from completing the training outlined in my initial Aims. A funding extension will allow me to complete these projects and gain critical training in 1) live time-lapse imaging to carefully examine BMP signaling dynamics in vivo, 2) chromatin-immunoprecipitation (ChIP) experiments to identify direct versus indirect targets of BMP signaling, and 3) co-immunoprecipitation (co-IP) experiments essential to identify the binding partners of the BMP target genes, Id1/2/3/4. This training is essential for me to gain the skills necessary to establish a strong independent research program that will make lasting impacts on the field of BMP signaling in craniofacial development.