Abstract Signaling molecules and their receptors rely on endo-lysosomal trafficking to prevent their accumulation on the plasma membrane, which would perturb craniofacial development. The ESCRT (Endosomal Sorting Com- plexes Required for Transport) multiprotein machinery is recruited to endosomal membranes for the degrada- tion of such endosomal cargoes. However, ESCRT functions during vertebrate development are poorly under- stood. We isolated a mouse line carrying an ENU-induced hypomorphic mutation of the ESCRT-II-encoding gene Vps25, Vps25ENU. Vps25ENU/ENU homozygous embryos exhibit polydactyly with hyperactivation of the fi- broblast growth factor (FGF)-sonic hedgehog (SHH) feedback loop. We uncovered that: 1) ESCRT-encoding genes, including Vps25, are expressed in both cranial neural crest (CNC)-derived mesenchyme and epithelium and enriched in select craniofacial domains; 2) Vps25ENU/ENU embryos exhibit severe craniofacial defects, in- cluding branchial arch 1 (BA1)-derived structures; 3) FGF and SHH signaling are unperturbed in Vps25ENU/ENU heads, unlike in limbs; 4) Vps25 cephalic epithelium- and CNC-specific loss, obtained using our new Vps25 conditional allele, phenocopy the craniofacial defects of Vps25ENU/ENU mutants; 5) select head domains of Vps25ENU/ENU embryos exhibit alterations of sphingolipid metabolism that are unaffected in mutant limbs; and 6) perturbations of lipid-synthesizing enzymes occur in both head epithelium and mesenchyme of Vps25ENU/ENU BA1, including upregulation of the Serine Palmitoyl Transferase (SPT) enzymatic complex critical for sphin- golipid synthesis. Thus, we hypothesize that the ESCRT machinery executes tissue-specific roles during mam- malian development by differentially regulating select signaling and metabolic pathways in discrete embryonic domains. We will address our hypothesis through these aims: 1) Determine whether the contributions of VPS25/ESCRT-II to mammalian craniofacial morphogenesis are tissue-specific. Using our conditional al- lele, we will assess whether VPS25 regulates the same or different signaling pathways in the murine cephalic epithelium and mesenchyme. 2) Characterize ESCRT-II-mediated dysregulation of lipid metabolism in Vps25-deficient embryos. We will assess tissue-specific lipid distribution and levels in Vps25ENU/ENU embry- onic heads versus limbs and will identify specific lipid species that are perturbed in Vps25-deficient heads by lipidomic analysis of BA1 tissue. 3) Establish if ESCRT-II-dependent perturbation of lipid metabolism is causative of the craniofacial defects of Vps25-deficient embryos. We will determine whether pharmaco- logic downregulation of SPT rescues, even partially, the craniofacial defects of Vps25 mutants. As SPT activity is negatively regulated by ORMDL proteins, we will assess in genetic interaction experiments if the head phe- notype of embryos with enhanced SPT activity due to loss of Ormdl3 on a Vps25ENU/+ background phenocopies that of homoz...