Figure 3. Temporal expression of retinal markers during differentiation (Days 9–43). A: Representative images from Experiments 2 and 3 showing expression of eyefield progenitor markers PAX6, SOX2, SIX3, CHX10, OTX2, LHX2 and RAX on D9. B: Immunofluorescence at D31 showed expression of OTX2 and CRX, with no detectable signal for OPSN-S or RHO. Expression of NR2F2, RCVRN, PRDM1, and TUJ1 was observed. C: At D36, expression of OTX2, CRX, OPSN-S, RCVRN, NR2F2, RxRg, PRDM1, and TUJ1 was evident. D: By D42, expression of CRX, OPSN-S, and RCVRN was maintained, along with LHX2. Some OTX2 expression persisted, and PRDM1 and TUJ1 remained detectable. E: At D43, immunocytochemistry revealed expression of RHO, OPSN-M/L, and RCVRN. Cell nuclei were counterstained with Hoechst 33342.