Fig 2: Comparison of Fetal Retinal Cultures (Retinospheres) with All Datasets(A) FD130 retina with fovea and optic nerve head (ON).(A’) Pieces of the retina prior to culture.(B) Age at culture of retinospheres used in this study. FD45+21 refers to a FD45 retina cultured for 21 days.(C) Retinospheres made from older retinas organized into spheres (D127+7, D137+13) and could be cultured for a month (D127+28) or up to 4 months withprominent outer segments (D127+112).(D and D’) Combined UMAP plot of retinoshperess, fetal retina, and organoids. (D’) shows UMAP plotted by tissue of origin: retinospheres (RSD76 [RSD, retinosphere age in days] cultured until day 109, RSD91 cultured until day 109, RSD78 cultured until day113), organoids (day 104 and day 110), and fetal retina (FD82 central and periphery).(E and F) Percent counts from (D) for (E) FD82 retina with equivalent age retinospheres (i.e., retinosphere cultured from D70 to D100) and D110 organoids highlightsimilar composition across all samples. (F) This is also true for older ages of fetal retina (FD125C+P) when compared to older cultures of retinospheres (RSD122 cultured for 14 days and RSD130 cultured for 30 days) and D205 organoids.(G–H’) Fovea-derived retinospheres lack rods (NR2E3, magenta, G and G’), unlike nasal central (NC)-derived retinospheres (H and H’).(I) Retinospheres maintained for 100+ days retain lamination, as seen by RCVRN+ PRs (magenta, ONL) and BCs (RCVRN, OTX2, and Goa).(J and K) RSD80 (K) resemble equivalent age organoids (D90, J) but demonstrate distinct and compact rings of the AC/RGC marker HUC/D.(L) At later ages, organoids express NRL-GFP, and the ONL is well preserved, but calretinin staining does not form a continuous layer.(M) After 134 days in vitro, retinospheres continue to maintain inner retinal layers (CALRET/HUC/D).Scale bars, 50µm.

Fig 3: Retinal differentiation of iPSCs generated on the Cell X platform. A Schematic of retinal differentiation protocol. Representative phase micrograph of day 0 iPSCs (B), day 7 EBs (C), day 16 attached optic vesicles (D), and day 55 lifted retinal organoids. Scale bar = 1 mm. Immunohistochemical staining of retinal organoids at day 120 (F– I) and day 160 (J–N). Antibodies targeted the photoreceptor cell markers OTX2 (green) and Recoverin (red), the rod photoreceptor cell markers NRL and NR2E3 (green) and cone photoreceptor cell marker ARR3 and ML-Opsin (red). DAPI (blue) was used as a nuclear counterstain

Fig 4: Activity of Nr2e3 cis-regulatory elements in mouse retinas. (A-D) Co-electroporation of one of the Nr2e3 elements driving an AP reporter or the control Stagia3 vector with CAG::mCherry (signal shown in A’-D’) to confirm electroporation in E13 mouse retinas. (E-H) Same DNA constructs as in A-D, but using P0 mouse retinas.(E’-H’) CAG::mCherry signal from retinas shown in E-H. (I-L) Sections of P0 retinas shown in E-H showing location of AP reporter-positive cells. (I’-L’) CAG::mCherry signal of sections shown in I-L. Scale bar in A is 100um and applies to A-D and A’-D’. Scale bar in E is 200 µm and applies to E-H and E’-H’. Scale bar in I is 20 µm and applies to I-L and I’-L’

Fig 5: Summary of the activity of cis-regulatory elements tested in this study. The elements shown in yellow are active in both cone and rod photoreceptors in a reporter assay. The CrxEnh1 and Gnb3Enh1 elements are also active in developing bipolar cells. The ThrbCRM1 and ThrbCRM2 elements (in green shading) are cone specific and the Rhodopsin element is rod specific in the assays used in this study. The Nr2e3 elements show a less clear segregation to cone or rod specific activity as illustrated in the summary of the assays shown below. ND is Not Determined. Dashed lines represent putative indirect effects