Generation of organoids from medaka pluripotent embryonic cells.

(a) Schematic representation of organoid generation. At day 0, pluripotent embryonic cells were extracted from blastula-stage medaka embryos and seeded in U-bottom, low-binding 96-well plate into DMEM/F12 media supplemented with 5 % KSR and 20 mM HEPES pH 7.4. At day 1 aggregates were supplemented with 2 % Matrigel. (b) Bright-field pictures of indicated stages of organoid formation showing the appearance of lens-like structure (asterisk). KSR, knockout serum replacement. Pictures acquired with wide-field microscope (Mi8; Leica). Scale bar 100 μm.

Fish retinal organoids form lenses.

(a) Expression of lens-specific markers (Cry::ECFP, lens fiber cell (LFC) and Prox1) and retina-specific marker (Atoh7::EGFP) in medaka embryonic eyes at 3 dpf. Optical sections showing the expression of indicated markers detected by whole-mount immunohistochemistry with the use of anti-GFP (green), anti-LFC (magenta) and anti-Prox1(cyan) antibodies, co-labelled with DAPI nuclear stain (grey). (b) Organoid generation from crystallin reporter line (Cry::ECFP). Expression of lens-specific markers (Cry::ECFP and LFC) in day 4 organoids detected by anti-GFP and anti-LFC antibodies. Represented by maximal projection for overview and single optical section for the detail of the lens. (c) Expression of Prox1 in day 4 organoids derived from wild-type medaka cells detected with anti-Prox1 antibody, co-stained with DAPI nuclear stain; displayed as maximal z-projection. (d) Organoid generation from retina-specific reporter line (Atoh7::EGFP). Overlay of bright-field and wide-field images showing EGFP fluorescence in Atoh7-expressing cells, lens indicated with asterisk. (e) Organoids generated from retina-specific reporter line (Rx3::H2B-GFP) labeled with anti-GFP and anti-LFC antibody displayed as maximal z-projection. (f) Schematic representation of distribution of retina- and lens-specific markers in day 4 organoids. (g) Optical section of day 4 organoid lens labeled with anti-Prox1 and anti-LFC antibodies, co-labeled with DAPI, showing elongated Prox1+/LFC+ cells on the surface of the lens. (h) Enlargement of indicated area in g. For Cry::ECFP, Atoh7::EGFP and Rx3::H2B-GFP. The percentage (%) indicates how many cells carry the indicated transgene. Arrow in b, c and e indicates position of the lens. Arrowheads in b and h indicate abnormally looking nuclei. le, lens; re, retina; wt, wild-type; LFC, lens fiber cell. Scale bar 50 μm in a, g and enlargement of b, 100 μm in b-e, 10 μm in h.

The lens originates from a spatially separated population of lens progenitors in center of the organoid.

(See also Figure S2 and S3, Video S1, S3 and S4.) (a) Schematic representation of lens formation in medaka. The lens develops from lens-competent head surface ectoderm (SE, magenta), retinal cells from the OV (green). Surface ectoderm forms the lens placode (lp) that further invaginates together with the OV to form the optic cup with a lens sphere surrounded by the retina. (b) Optical sections through wild-type medaka embryos at indicated stages stained with the antibody against Prox1, a lens placode-specific marker and LFC, a lens fiber cell marker. (c) Optical sections through organoids derived from the Rx3::H2B-GFP reporter line stained with antibody against Prox1 (magenta) and LFC (cyan). (d) Lens specific GFP expression in embryos of the Foxe3::GFP reporter line (magenta). Bright-field and GFP fluorescence images showing reporter expression in presumptive lens ectoderm (ple) of day 1 (S19), lens placode (pl) of 1 dpf (S21) and lens (le) of 2 dpf (S24) medaka embryos. (e) Optical section through a day 1 organoid (26 hpa) derived from Foxe3::GFP reporter line showing the lens-specific expression of GFP (magenta) detected by anti-GFP antibody, co-stained with DAPI (blue). (f) Optical section through a day 2 organoid derived from 50 % Foxe3::GFP and 50 % Rx2::H2B-RFP reporter blastula cells. Expression of RFP (green) and GFP (magenta), detected by antibodies is mutually exclusive, co-stained with DAPI (blue). (g) 3D rendering of Rx2::H2B-RFP (green), Foxe3::GFP (magenta) organoid shown in f, DAPI stained nuclei are in cyan. n numbers in c and e indicate the number of analyzed organoids with described phenotype. LFC, lens fiber cell; le, lens; re, retina; lp, lens placode; SE, surface ectoderm; OV, optic vesicle; dpf, day post fertilization; hpa, hours post aggregation. Dashed line in c, e and f indicates the outline of the organoid. Dashed line in d indicates the outline of the retina. Scale bar 100 μm.

Dynamics of lens formation in ocular organoids.

(a) Single organoid (from Video S2) generated from Foxe3::GFP reporter (50 %) and wild-type blastula cells imaged from day 1 (24 h post aggregation; hpa) to day 2 (40 h post aggregation; hpa). Overlay (top panel) of bright-field and GFP fluorescence and GFP fluorescence (bottom panel, gray) of indicated timepoints between day 1 and day 2 organoid development. Dashed lines indicate the outline of the organoid. Total number of n=62 organoids in 4 independent experiments were followed. (b) Onset of GFP expression in Foxe3::GFP-derived organoids grown with or without Matrigel supplementation at day 1, n=8 organoids per condition. Statistical significance was calculated using an unpaired two-tailed t-test. n.s., not significant. (c) Tracking of individual GFP+ cells in the organoids derived from mixture of Foxe3::GFP (10 %) and wild-type (90 %) cells from day 1 to day 2. Time indicated in hours post aggregation (hpa). (d) Tracks of individual cells over time from details 1-4 in c. (e) Movement-associated cell shape changes of GFP+ cells in in the organoids derived from mixture of Foxe3::GFP (10 %) and wild-type (90 %) cells from day 1 to day 2. Scale bar 100 μm; enlargement in e: scale bar 25 μm.

Lens size scales with organoid size.

(a) Schematic representation of generation of organoids of different sizes. Pluripotent embryonic cells were seeded in different seeding densities. (b) Representative optical sections of day 1 organoids generated by aggregation of indicated number of cells from Rx3::H2B-GFP reporter line immunolabeled with anti-GFP antibody, co-stained with DAPI. For Rx3::H2B-GFP, the percentage (%) indicates how many cells carry the indicated transgene. (c) Representative images of day 4 organoids generated by indicated number of cells stained with an anti-LFC antibody (maximal z-projections). (d) Quantification of diameter of day 1 organoids generated by aggregation of indicated number of cells. (e) Quantification of depth of retina-specific marker expression (Rx3::H2B-GFP) at day 1 generated by aggregation of indicated number of cells. Number of analyzed organoids: n=3 for 4,000 cells, n=8 for 2,000 cells, n=7 for 1,000 cells, n=9 for 500 cells. (f) Quantification of lens size measured as diameter of the lens in day 4 organoids. n=7 for 4,000 cells, n=13 for 2,000 cells, n=19 for 1,000 cells, n=19 for 500 cells. (g) Efficiency of lens formation in organoids of different sizes measured as proportion of organoids carrying lens judged by immunolabeling with anti-LFC antibody. Statistical significance was calculated using an unpaired two-tailed t-test. n.s., not significant; ****p<0.0001, *p<0.05. le, lens; re, retina; LFC, lens fiber cell. Scale bar 100 μm.

Lens formation is dependent on temporal activity of FGF and BMP signaling.

(a) Activity of BMP signaling detected by immunohistochemistry against phosphorylated SMAD1/5/8 (pSMAD1/5/8) in day 1 organoids. (b) Activity of FGF signaling detected by immunohistochemistry against phosphorylated ERK1/2 (pERK1/2) at indicated stages of organoid development and in embryo at 2 dpf. Dashed line indicates the outline of the organoid in day 1 and day 1,5 organoid. In day 2 organoid, line outlines the lens. n numbers indicate the number of organoids with displayed phenotype within one experiment. (c) Schematic representation of organoid treatment with FGF and BMP signaling antagonists. Organoids were generated from Foxe3::GFP reporter line. Inhibitors of BMP (Noggin, 100 ng/μl or Dorsomorphin -DM, 100 ng/ml) or FGF (SU5402, 10 μM) signaling were administered in time window from day 0 to day 1 or from day 1 to day 2. Organoids were analyzed at day 2 for the expression of lens progenitor marker Foxe3::GFP and lens fiber cell differentiation marker LFC. (d) Representative images of day 2 organoids treated as indicated and labeled with antibodies against GFP (green), LFC (magenta) and Rx2 (grey). Overview images are represented by maximal z-projections; single organoids are represented by single optical sections. For Foxe3::GFP, the percentage (%) indicates how many cells carry the indicated transgene. (e-f) Quantification of expression of Foxe3::GFP and LFC in control and treated organoids measured as proportion of organoids expressing lens progenitor marker Foxe3::GFP (green) and lens differentiation marker LCF (magenta). Total number of n=204 organoids grouped in control and treated groups were analyzed. All treatments were performed in two biological replicates. re, retina; le, lens; DM, Dorsomorphin; LFC, lens fiber cell. Scale bar 100 μm.

Lens organoid gene expression recapitulates gene expression in vivo.

(a) Schematic representation of sample preparation. Whole organoids (composed of both lens and retinal tissue) were analyzed. For 3 dpf and 4 dpf medaka embryos, eyes were dissected (composed of both lens and retina). For 1 dpf and 2 dpf embryos the anterior part of the head right behind optic vesicles or optic cup, respectively was dissected and used for RNA isolation. (b) Principal component analysis of the transcriptomes of embryonic and organoid samples at the indicated stages. (c) Heatmap clustering of variance stabilized counts of lens-specific genes. Samples were processed in 3 technical replicates each. blast, blastula-stage embryo; emb, embryo; lens_org, lens organoid.

Expression of lens-specific markers is dependent on the presence of HEPES in the media and occurs independently of Matrigel addition, related to Figure 1 and Figure 2.

(a) Day 2 organoids generated in DMEM/F12 + 5 % KSR media in the presence or absence of 20 mM HEPES labeled with anti-LFC antibody, co-labelled with DAPI nuclear stain. (b) Quantification of the efficiency of lens formation measured by the expression of lens marker LFC in day 2 organoids grown in differentiation media with or without HEPES in n=6 independent experiments (e1 – e6). Total number of analyzed organoids: n=63 without HEPES; n=83 with HEPES. (c-f) Impact of Matrigel supplementation on organoid organization. (c) Day 2 organoids supplemented or not supplemented with Matrigel (2 % final concentration) at day 1 labeled with anti-Rx2 and anti-AcTub (acetylated tubulin) antibody to visualize the structure of retinal neuroepithelium. (d) Day 2 and day 5 organoids supplemented or not supplemented with Matrigel (2 % final concentration) at day 1 labeled with anti-LFC antibody (displayed as maximal projections of multiple z planes). (e) Quantification of the size of the lens measured as the diameter of LFC+ lens sphere in d. Statistical significance was calculated using an unpaired two-tailed t-test. n.s., not significant. (f) Day 4 organoids labelled with anti-GFP and anti-LFC antibody, co-labeled with DAPI nuclear stain, represented by single optical section. The effect of Matrigel supplementation was assessed in total of n=49 organoids in 4 independent experiments. n numbers indicate the number of organoids with displayed phenotype within one experiment. LFC, lens fiber cell; re, retina; le, lens. Scale bar in a, c, d, and f: 100 μm, in enlarged lens in f: scale bar 50 μm.

Expression domain of Rx3::H2B-GFP in the embryo and organoid, related to Figure 3.

(a) Expression domain of Rx3::H2B-GFP in medaka fish embryo at indicated stages showing the expression in the anterior neural plate - presumptive eye field (S16, late gastrula stage, arrowhead), retina-committed progenitors of the optic vesicle (1 dpf), retina and forebrain (2 dpf). 1 dpf is represented by maximal z-projection. 2 dpf represented by single optical section through a live embryo. (b) Optical section through medaka fish embryo 1.5 dpf (S22) immunostained with antibodies against GFP and Prox1 showing lens-specific expression of Prox1 and retina-specific expression of Rx3::H2B-GFP. (c) Schematic representation of organoid generation from the retina-specific Rx3::H2B-GFP reporter line (50 % of cells). (d) Single optical section showing peripheral expression of retina specific marker Rx3::H2B-GFP in the organoid at day 1 visualized by immunohistochemistry with antibody against GFP; co-stained with DAPI nuclear stain. For Rx3::H2B-GFP, the percentage (%) indicates how many cells carry the indicated transgene. Dashed line indicates the outline of the organoid. wt, wild type; le, lens; re, retina; OV, optic vesicle. Images acquired with laser scanning confocal microscope sp8 (Leica). Scale bar 100 μm.

Characterization of Foxe3::GFP reporter-expressing cells in embryos and organoids, related to Figure 3.

(a-b) Expression of Foxe3::GFP in medaka embryonic lens. Optical sections of the whole eye and magnification of lens at 3 days post fertilization (a) and lens at 4 days post fertilization (b) showing the expression of Foxe3-driven GFP in lens epithelium (arrows) and differentiating lens fiber cells (core of the lens), co-labeled with the antibody against Prox1 and LFC lens fiber cell antibody. Dashed line indicates the outline of the lens in a. (c) Expression of Rx2::H2B-GFP and Foxe3::GFP, in the day 2 embryo and organoid. (d) EdU incorporation assay labeling proliferating (S-phase) cells in day 2 Foxe3::GFP-derived organoids exposed to EdU for 1 hour, GFP (magenta) and EdU (cyan) were detected by immunolabeling with anti-GFP antibody and EdU detection. (e) Distribution of Foxe3-driven GFP and lens differentiation marker LFC. Dashed line indicates the outline of the organoid. (f) Detail of indicated area in e highlighting Foxe3::GFP+/LFC- cells, similar to the progenitor-carrying lens epithelium in embryonic lens (arrows). Dashed line indicates the outline of the lens. For Foxe3::GFP and Rx2::H2B-RFP, the percentage (%) indicates how many cells carry the indicated transgene. le, lens; re, retina; epi, lens epithelium; LFC, lens fiber cell; dpf, days post fertilization. Scale bar 100 μm.

Retinal and lens progenitor cells show differential adhesive properties allowing for cell identity-based cell sorting.

(a) Schematic representation of dissociation/re-aggregation experiment. Day 1 organoids derived from Foxe3::GFP lens reporter line were dissociated into single-cell suspension and left to re-aggregate. (b) Representative picture of day 1 Foxe3::GFP organoid before dissociation showing lens-specific GFP expression in the central region of the organoid. (c) Single cell suspension generated by trypsin-mediated organoid dissociation. (d) Process of re-aggregation showing gradual sorting of Foxe3::GFP-expressing lens progenitors. Time (t) indicated in hours post dissociation. (e) Representative images of organoids generated by dissociation/re-aggregation process showing distribution of retinal (AcTub+) and lens (GFP+) cells analyzed by immunohistochemistry. In 3 independent experiments, total number of n=35 organoids were analyzed. n numbers in e indicate the number of organoids with displayed phenotype within one experiment. Scale bar 100 μm.