Differentially expressed genes between neural crest and neural crest-derived clusters from Tg(mitfa:GFP) mitfa+/w2 and Tg(mitfa:GFP) mitfaw2/w2 embryos

PAX3 positive melanocytes are observed in mouse and human eyes by scRNA-Seq analysis

Immunofluorescence of the choroid using an anti-GFP antibody

Findallmarkers of the casper TEAZ-Eye single-cell object

Differentially expressed genes between BRAFV600E-driven tumors and GNAQQ209L-driven tumors in adult zebrafish

Findmarkers for choroidal melanocytes, skin melanocytes, TEAZ-Eye, and TEAZ-Skin versus their respective microenvironments; gene names in every intersection of the Venn Diagram comparison of all four groups

Oncogenic GNAQ expression in wild-type and casper eye tumors

Differentially expressed genes between eye and skin melanocytes and between TEAZ-Eye and TEAZ-Skin, respectively

Differentially expressed genes between cancer associated fibroblasts from wild-type TEAZ-Eye and normal fibroblasts from wild-type normal eyes

Immunofluorescent staining for Pax3 in the mouse limbus

CellChat analysis of intercellular communication between zebrafish fibroblast, CAF, and UM cell clusters

Abstract 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 preclinical animal models. In this study, we developed a primary, immunocompetent zebrafish model to study uveal melanoma 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 uveal melanoma pathogenesis. Significance: Choroid-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.

Melanocyte rescue experiments in nacre zebrafish by TEAZ-Eye using plasmids containing mitfa:mitfa; mitfa:GFP (miniCoopR)

Enamel, the hardest mineralized material in the human body, protects the underlying living tissues, the dentin and pulp of the tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel-producing cells, ameloblasts. iPSC-derived secretory Ameloblasts (isAM) have promise in future regenerative dentistry. Today, it is not known why iAM maturation requires intimate contact with the dentin-producing cell type, odontoblast. Here, we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. Notably, soluble Notch agonist induces the iAM maturation to a novel, WDR72-positive mature secretory AM stage (ismAM) in our ameloblast organoid model. When transplanted under the kidney capsule of NOD-SCID mice, these ismAM organoids generated enamel-like calcified material, as confirmed by microCT analysis, marking the first demonstration that Notch-activated iAM organoids can form such tissue in vivo. This novel maturation procedure enabled us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, a gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in human ameloblasts for the expression of Enamelin, MMP20, and WDR72, a role not previously demonstrated in mouse models.

Abstract Most heritable risk for orofacial clefts (OFC) remains unassigned to specific genes or loci. IRF6 and GRHL3 , two established OFC risk genes, encode transcription factors (TFs) essential for the differentiation of the periderm, a transient embryonic tissue required for secondary palate fusion. To identify novel risk candidates, we modeled the zebrafish periderm transcriptional regulatory network (TRN). Using single-cell multiome sequencing (RNA-seq and ATAC-seq) from shield-stage embryos, we inferred TF-to-target gene connections by integrating correlated gene expression with TF binding site predictions within chromatin elements open in periderm cells. We generated sets of gold-standard edges by conducting RNA-seq on TF-depleted embryos and ChIP-seq/CUT&RUN on wild-type embryos and used them to benchmark model performance. Within the top-performing model, zebrafish periderm modules are strongly preserved in human embryonic periderm and orthologs of human OFC-associated genes have higher centrality and edge-sum scores than non-associated genes. Functional validation confirmed the network’s predictive power: depleting high-centrality TFs, including grhl1 , klf6a , tead3b , and klf17 , disrupted periderm differentiation in sensitized embryos. Moreover, analysis of whole-genome sequencing data from 2,415 OFC trios identified 15 individuals with rare or de novo GRHL1 variants, four of which introduced premature stop codons. This study establishes GRHL1 as a novel OFC risk gene and highlights the power of cross-species gene regulatory network analysis to prioritize candidates for rare variants in complex structural birth defects.

Mitfa-deficient zebrafish have decreased tumor latency compared to wild-types by TEAZ-Skin

Ingenuity Pathway Analysis of genes enriched in TEAZ-Eye compared to the tumor microenvironment

Findallmarkers of the integrated object between wild-type control eyes and wild-type TEAZ-Eye

During skin development, ectoderm-derived cells undergo precisely coordinated proliferation, differentiation, and adhesion to yield stratified epidermis. Disruptions in these processes can result in congenital anomalies, including ectodermal dysplasia and harlequin ichthyosis. PRMT5-an enzyme responsible for methylating arginine residues in histones and other proteins-maintains progenitor status in germ and limb bud cells. Similarly, in vitro evidence suggests that PRMT5 prevents differentiation of basal keratinocytes, leading us to hypothesize that PRMT5 preserves the stem-cell phenotype of keratinocytes in vivo. To test this possibility, we generated conditional knockout mice lacking Prmt5 in early ectoderm (embryonic day 7.5), impacting the entire epidermis. Prmt5 conditional knockouts exhibited gross skin defects, compromised skin barrier function, and reduced postnatal viability. Histological analyses revealed significant defects in epidermal stratification, without alterations in apoptosis or proliferation. Single-cell RNA sequencing and Assay for Transposase-Accessible Chromatin with high-throughput sequencing analysis identified an atypical population of basal keratinocyte-like cells in Prmt5 conditional knockouts that exhibited a senescence-like program, characterized by increased Cdkn1a (p21), elevated senescence-associated secretory phenotype molecules (Igfbp2), and decreased developmental transcription factor (Trp63) expression. Our findings suggest that PRMT5 prevents basal keratinocyte senescence by repressing Cdkn1a, shedding light on the epigenetic regulation of basal keratinocyte maintenance and senescence in congenital skin disorders.