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). Optical section showing the overlay of bright-field and GFP fluorescence in the 1 dpf and 2 dpf medaka embryos (left and middle). Detail of the eye (right) showing the expression domain of Foxe3::GFP (magenta) and LFC (cyan) detected by immunohistochemistry. (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.

Single organoid (from Video S2) generated from Foxe3::GFP reporter (50 %) and wild-type blastula cells imaged from day 1 (24 h post aggregation; t=0 h) to day 2 (40 h post aggregation, t=16 h). 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. Scale bar 100 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) 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. (b) 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. (c) 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). 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. blast, blastula-stage embryo; emb, embryo; lens_org, lens organoid.