Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

Melanocytes reside in diverse microenvironments that influence their susceptibility to oncogenic transformation; however, investigation of rare melanoma subsets has been limited by the lack of suitable pre-clinical animal models. Here, we developed a primary, immune-competent zebrafish model to study uveal melanoma (UM) using choroidal melanocyte-targeted injection and electroporation of plasmids encoding human GNAQQ209L together with CRISPR/Cas9 cassettes for somatic tumor suppressor gene deletion. Single-cell transcriptional profiling of primary melanocytes and melanoma derived from the eye and skin revealed distinct transcriptional programs, with epithelial-to-mesenchymal transition pathways enriched in ocular tumors. In addition, choroidal fibroblasts from tumor-bearing eyes exhibited marked transcriptional changes, including increased fibronectin and collagen expression, consistent with stromal remodeling. Given prior associations between mitfa loss and accelerated GNAQQ209L tumor onset, the model was applied to determine whether melanocyte differentiation state contributes to the emergence of GNAQ-driven tumors. The increased susceptibility resulted from expansion of Mitfa-independent melanocyte progenitor populations in germline mitfa mutant zebrafish, rather than somatic mitfa loss in differentiated melanocytes, as conditional, melanocyte-specific mitfa deletion in adult zebrafish did not accelerate tumor growth. Furthermore, pax3a-positive melanocyte progenitor cells in mitfa-deficient zebrafish embryos and adult eyes and skin were highly susceptible to transformation induced by GNAQQ209L but not BRAFV600E. Analogous PAX3 positive populations were also identified in mouse and human single-cell transcriptomic datasets. Collectively, these findings establish a critical role for Mitfa-independent melanocyte progenitors in UM pathogenesis. Statement of SignificanceChoroid-targeted GNAQQ209L expression induces anatomically correct uveal melanoma in adult zebrafish, with germline mitfa deletion expanding mitfa-independent melanocyte progenitors with enhanced susceptibility that are transcriptionally distinct from the subpopulation transformed by BRAFV600E.