(a) Scheme representing stages and timing of the medaka fish retinal development. The retinal domain is indicated in green. Establishment of the eye field within the anterior neural plate is followed by the formation of optic vesicles at 1 day post-fertilization (dpf). Optic vesicle evagination is followed by the morphogenesis of a bi-layered optic cup formed by retina surrounded by retinal pigmented epithelium (RPE) and subsequent onset of retinal differentiation at 2 dpf. By 4 dpf, the major retinal cell types – retinal ganglion cells (yellow), amacrine cells (orange), bipolar cells (red), horizontal cells (cyan), and photoreceptor cells (blue) – are generated. (b) Schematic representation of aggregate generation, its timeline and culture conditions. At day 0, primary pluripotent cells were harvested from blastula-stage medaka embryos and re-aggregated in low binding U-shape 96-well plates. At day 1, the culture media was supplemented with Matrigel. At day 2, the aggregates were transferred to a low binding culture plate and maintained in 3D suspension culture conditions in DMEM/F12 media supplemented with 5% FBS, 5% FEE, and N2 supplement. The gross morphology of the aggregates was analyzed at days 1, 2, and 3. KSR, knockout serum replacement; FBS, fetal bovine serum; FEE, fish embryonic extract. (c) Dark-field images of a blastula-stage embryo, a blastula-derived cell suspension, and re-aggregated cells and the gross morphology of aggregates at days 1, 2, and 3 after re-aggregation. (d) Optical section showing aggregate organization at day 2 visualized by immunostaining against neuroepithelium-specific markers, N-cadherin (Ncad), and acetylated tubulin (AcTub), co-stained with DAPI nuclear stain. Scale bars: 100 and 50 μm (enlargement in (d)).

Bright-field images of day 1 aggregates derived from early morula (64–128 cells), late morula (256–512 cells), and early blastula (1000 cells). Scale bar: 100 μm.

Virtual planes of four day 2 aggregates, stained with N-cadherin (Ncad) and DRAQ5 nuclear stain, showing the interior cellular composition using SPIM imaging. Scale bar: 100 μm.

(a) Schematic representation of the Rx3::H2B-GFP transgenic construct and the corresponding expression domain of GFP in the optic vesicles of a developing medaka embryo at 1 dpf. (b) Scheme of organoid generation from Rx3::H2B-GFP transgenic fish. (c) Bright-field and fluorescence images of aggregates derived from Rx3::H2B-GFP transgenic fish at 0.5, 6, and 16 hpa. (d) Analysis of the onset of GFP expression in Rx3::H2B-GFP-derived fish embryos (n = 6) and organoids (n = 17). ***p<0.001. (e) Optical sections of day 1 (before the addition of Matrigel) and day 2 organoids derived from Rx3::H2B-GFP transgenic fish stained with antibodies against N-cadherin (Ncad) and GFP, co-stained with DAPI nuclear stain. (f) Generation of Rx3KO (Rx3saGFP) line – schematic representation of the Rx3 locus with integrated saGFP-OPT cassette. An open reading frame-adjusted gene trap cassette comprising a splice acceptor and a GFP sequence (saGFP) followed by a polyA and a strong terminator sequence derived from the ocean pout (OPT; STOP) were inserted into the Rx3 locus. (g) Scheme of organoid generation from Rx3-deficient single blastulae. (h) Bright-field and fluorescence images of phenotypes of Rx3saGFP/+(Rx3 +/- heterozygote) and Rx3saGFP/saGFP (Rx3 -/- homozygote) mutants at 1 dpf and corresponding organoids at day 2. dpf, days post-fertilization; hpa, hours post-aggregation; hpf, hours post-fertilization; wt, wild type. Scale bar: 100 μm.

(a) Schematic representation of primary embryonic pluripotent cell culture. Blastula-stage embryos were dissociated and cultured either under rocking suspension culture condition or as individual cells in wells of 96-well plates. (b) Overlay of bright-field and fluorescence images of cells derived from Rx3::H2B-GFP transgenic fish and cultured for 24 hr under rocking suspension culture conditions. Magnification shows three individual clusters of cells. Dashed line outlines the borders of an individual cluster. (c) Overlay of bright-field and fluorescence images of cells derived from Rx3::H2B-GFP transgenic fish cultured individually in wells of 96-well plates at time 0.5 hr (just about 30 min after dissociation and seeding) and 19 hr of the culture. Scale bars: 100 μm.

Bright-field and fluorescence images of day 2 organoids derived from Rx3::H2B-GFP transgenic fish incubated with and without Matrigel from day 1 to day 2. Membrane stain (CellMask) shows the organization of the neuroepithelium. Scale bars: 100 and 50 μm (enlargement in detail of neuroepithelium).

Optical sections of embryonic heads (upper panel) and organoids (lower panel) stained with antibodies showing expression of Rx2, Sox2, and Lhx2 co-stained with DAPI nuclear stain. Dashed lines indicate optic vesicles in an embryo or neuroepithelium in an organoid. dpf, days post-fertilization. Scale bar: 100 μm.

Bright-field images of fish embryos 4 dpf showing the phenotypical consequence of cassette integration in the Rx3 locus. dpf, days post-fertilization. Scale bar: 100 μm.

(a) Scheme of aggregate generation from Rx3::H2B-GFP DNA-injected blastulae. (b) Bright-field images of zebrafish-derived aggregates at 1 hpa and 1 dpa. (c) Analysis of the onset of GFP expression in Rx3::H2B-GFP-derived zebrafish embryos (n = 6) and organoids (n = 15). ^**p<0.01. (d) Fluorescent images of mCherry and GFP expression domains in Rx3::H2B-GFP-injected embryos at 1 dpf and corresponding aggregates at day 1. Dashed lines indicate the embryonic retina and organoid retinal neuroepithelium, respectively. wt, wild type; dpf, days post-fertilization; dpa, days post-aggregation; hpa, hours post-aggregation. Scale bar: 100 μm.

(a) Fluorescence and bright-field images of day 2 organoids produced by aggregation of >1000 cells and <1000 cells stained with anti-Rx2 antibody. (b) Analysis of the area of Rx2 (wild-type organoids stained with anti-Rx2 antibody) and Rx3 (Rx3::H2B-GFP-derived organoids stained with anti-GFP antibody) expression area (% of total organoid area) in day 2 organoids. ****p<0.0001. (c) Number of organoids forming (1–4) individual retinal regions produced by aggregation of >1000 (n = 26 for Rx2, n = 9 for Rx3) and <1000 (n = 57 for Rx2, n = 66 for Rx3) cells from nine independent experiments. (d) Size, measured as largest circumference, of the optic vesicle of 1 dpf embryos and optic vesicle-like structures formed by day 2 organoids (n = 16 embryos, n = 56 aggregates from six independent experiments). ns, non-significant. (e) Bright-field and fluorescent images of day 2 Rx3::H2B-GFP organoids stained with anti-GFP antibody. Optical sections of an organoid (day 2) (n = 9/10) and an embryo (1 dpf) stained with anti-Rx2 and anti-Otx2 antibodies. (f) Maximal projection of day 2 organoids and 1 dpf embryo generated from Rx3::H2B-GFP transgenic or wild-type blastulae and stained with neural tissue-specific anti-Sox2 (n = 12/12) and anti-Otx2 (n = 10/10) antibodies, co-stained with anti-Rx2 and DAPI nuclear stain. hpa, hours post-aggregation; dpf, days post-fertilization. Scale bars: 100 μm.

Overlay of fluorescence and bright-field images of retinal organoids on day 2 generated from the Rx3::H2B-GFP reporter line formed by aggregation of <1000 primary embryonic pluripotent cells stained with anti-GFP antibody. Scale bar: 100 μm.

Fluorescence images, bright-field images, and their overlay of retinal organoids on day 2 generated from the Rx3::H2B-GFP reporter line formed by aggregation of <1000 primary embryonic pluripotent cells stained with anti-GFP antibody. Scale bar: 100 μm.

(a) In vivo time-lapse images acquired with MuVi SPIM of optic vesicle-like structure evagination in Rx3::H2B-GFP retinal organoids. From top to bottom: maximal projection of quasi bright field, GFP, and tracks of exemplary cells. (b) Normalized velocity autocorrelation for all tracks (n = 4600; from two optic vesicles of one organoid). (c) Directional histogram of tracks (n = 4600; from two optic vesicles of one organoid) separated for inward and outward movements. hpa, hours post-aggregation; dpf, days post-fertilization. Scale bar: 100 μm.

(a) Scheme of organoid generation from Atoh7::EGFP transgenic fish. (b) Fluorescent images of day 3 Atoh7::EGFP organoids (generated by aggregation of <1000 cells). (c) Optical sections and maximal projections showing EGFP expression in the eye of the developing embryo at 2 dpf and the retinal organoid at day 3 co-stained with antibody against HuC/D and DAPI. (d) Optical sections showing expression of HuC/D (amacrine and ganglion cells), Otx2 (bipolar and photoreceptor cells), and Prox1 (horizontal cells) in day 4 organoid. (e) Sketch of the arrangement of cellular layers in the organoid and the embryonic retina. dpf, days post-fertilization. Scale bar: 100 μm.

(a) Scheme of organoid generation from Atoh7::EGFP transgenic fish. (b) Bright-field and fluorescent images of day 3 Atoh7::EGFP organoids. (c) Optical sections of day 4 Atoh7::EGFP organoids co-stained with antibodies against HuC/D (amacrine and ganglion cells), Otx2 (bipolar and photoreceptor cells), and Prox1 (horizontal cells). Dashed lines indicate the retinal domain. Scale bar: 100 μm.

(a) Schematic representation of CHIR-99021 treatment. Day 1 aggregates were washed and incubated with Matrigel for 6 hr. Organoids were washed and incubated in media containing 5 μM CHIR-99021 or DMSO. (b) DMSO- and CHIR-99021-treated day 2 organoids and medaka embryonic eye (42 hr post-fertilization [hpf]) showing expression of retina- and RPE-specific transcription factors Rx2 and Otx2, respectively. (c) DMSO- (n = 6/6) and CHIR-99021-treated (n = 13/13) day 4 organoids, including section through CHIR-99021-treated organoid stained with anti-Otx2 antibody. Dashed lines indicate retinal or RPE domains. re, retina; le, lens; RPE, retinal pigmented epithelium. Scale bars: 100 μm, 50 μm in enlargements in b and in fish embryonic retina in b.