**Background:** The retinal pigmented epithelium (RPE) is a critical tissue for eye development and retinal function, originating from neural progenitors of the anterior neural tube. Its differentiation involves a gradual process of pigmentation, polarity acquisition, and expression of visual cycle genes, regulated by transcription factors (TFs) such as MITF, OTX2, PAX6, and SOX9. Chromatin remodeling complexes, particularly SWI/SNF (BAF), are known to regulate TF activity by modulating chromatin accessibility, but their specific roles in RPE differentiation were unexplored. This study aimed to characterize the spatial transcriptome of the developing RPE and determine the role of SWI/SNF complexes in RPE maturation and the suppression of neural and proliferative programs.
**Methods:** The authors used a mouse model with conditional inactivation of both Smarcc1 and Smarcc2 (encoding BAF155 and BAF170 subunits) specifically in pigmented epithelial (PE) progenitors via DCT-Cre (FcKO). At embryonic day 14.5 (E14.5), a stage of ongoing RPE maturation, they performed geographical position sequencing (Geo-seq) on laser-capture microdissected samples from eight positions along the proximal-distal and dorsal-ventral axes of the optic cup from both PE and adjacent ocular mesenchyme (Me). Four control and three FcKO eyes were analyzed, yielding a mean depth of 30 million reads per sample. Differential expression analysis (DESeq2) identified 4297 DEGs between PE and Me in controls, and 2693 unique DEGs in FcKO vs. control PE. Regulon analysis (SCENIC) identified 173 regulons. Cell proliferation was assessed by EdU pulse labeling and MKI67 immunostaining, and protein expression was validated by immunofluorescence.
**Key Results:** In control PE, Geo-seq revealed spatial gradients of gene expression: proximal regions (O, CO) expressed maturation genes (e.g., Sox9, extracellular matrix, fatty acid metabolism), while distal regions (CT, T) expressed proliferation and glycolysis genes (e.g., Pax6, Mcm3-7). Regulon analysis identified key TFs: MITF (1261 targets), BHLHE41 (1062 targets), SOX9 (111 targets), SOX10 (448 targets), PAX6 (139 targets), and OTX2. In FcKO PE, SWI/SNF inactivation caused hypopigmentation and thickening of the PE layer. Transcriptomic analysis showed downregulation of 1182 genes (∼80% of DEGs) enriched for pigmentation, lysosome organization, and extracellular matrix, and upregulation of 1206 genes enriched for cell proliferation (e.g., Bub1, Cenpe, Mki67) and neural genes (e.g., Neurog2, Sox11, Ascl1, Pou3f2, Tubb3, Map2, Nefm). Cell-cycle scoring classified 87.5% of FcKO samples as G2/M phase vs. ∼50% of controls as G1. EdU and MKI67 quantification confirmed a significant increase in proliferating cells in all PE regions of FcKO (e.g., proximal O: control EdU+ ∼10%, FcKO ∼40%; p<0.05). Immunofluorescence showed loss of SOX9 and OTX2 proteins, maintained MITF protein, and ectopic expression of PAX6, CDH2, TUBB3, and MAP2 in FcKO PE, with some neural markers co-expressed with MKI67. Regulon analysis revealed reduced activity of SOX9, SOX10, OTX2, and MITF regulons, and increased activity of PAX6, LEF1, E2F2, and neural TFs (SOX11, POU3F2).
**Clinical Implications:** This study provides the first high-resolution spatial transcriptomic atlas of the developing mouse RPE and identifies SWI/SNF complexes as essential regulators of RPE differentiation. The findings demonstrate that SWI/SNF complexes are required for the expression and activity of key RPE TFs (SOX9, OTX2, MITF) while simultaneously repressing cell proliferation and neural differentiation programs. The observation that SWI/SNF-deficient PE cells express neural progenitor genes but not retina-specific markers suggests a partial transdifferentiation, highlighting the complexes' role in maintaining RPE identity. These insights have implications for understanding RPE-related diseases (e.g., age-related macular degeneration, inherited retinal dystrophies) and for regenerative medicine, as modulating SWI/SNF activity could potentially influence RPE plasticity and repair. The spatial transcriptomic data (available online) serve as a valuable resource for future studies on RPE development and disease.