Figure 2. Stage-specific transcription factor and gene expression during retinal differentiation. A: Immunofluorescence analysis revealed dynamic expression of transcription factors and retinal markers across differentiation stages. At D6, pluripotency markers OCT4 and SOX2 were detected. During primary differentiation, the retinal neurogenesis markers RAX, PAX6, and LHX2 emerged. By D30, expression of the neural retinal progenitor cell (NRPC) marker CHX10 and photoreceptor (PhR) subtype-specifying marker NR2F2 was observed. At D46, RCVRN and NR2F2 were present alongside diminishing CHX10, with sustained expression of RAX and PRDM1. By D66, OPSN-M/L and RCVRN were detected. Cell nuclei were counterstained with Hoechst 33,342. B: RT-qPCR analysis of gene expression across five time points (D0, D6, D30, D40, and D59) during the differentiation protocol. Expression levels were normalized to GAPDH and presented as percentage of maximal expression for each gene. The data show expression then downregulation of pluripotency gene OCT4, followed by upregulation of eyefield gene OTX2, NRPC gene VSX2, and early PhR lineage markers PRDM1, CRX, RCVRN, and NR2F2.