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1 doi: /nature09941 Supplementary Figures and legends Supplementary Fig. 1 Eye-cup formation in vivo and in ESC culture. a, Schematic of in vivo retinal development. b, SFEBq culture for retinal induction. c, FACS analyses for differentiation of Rx::GFP + retinal progenitors in SFEBq culture with matrigel on day 7. d-f, Efficient induction of Rx::GFP + retinal tissues in SFEBq culture using 1.5% Knockout Serum Replacement (KSR)-containing medium and matrigel (MG) (d). Inhibition of Rx induction by treating ES cells with the 1

2 anti-integrin-beta1 antibody from day 1 (e). Induction of Rx::GFP + retinal tissues in SFEBq culture either with 2% matrigel (f, lane 2) or with purified laminin (LN) + entactin (EN) and Nodal (day 1-7) (f, lane 4). Inhibition by 10 µm SB (SB; Nodal/Activin inhibitor). g, ESC aggregate on day 1 uniformly immunostained for the undifferentiated-cell marker Oct3 and Nanog. h-j, Spontaneous formation of N-cad + neuroepithelium in the SFEBq aggregate on day 5. See the strong N-cad + staining (inner) indicating the apical side of the neuroepithelium (h). Immunostaining of the apical (N-cad, CD133) and basal (laminin) markers in day-5 culture (i-j). k, An example of SFEBq aggregates carrying four Rx::GFP + optic cups. l, Rx::GFP + eye-cup formation in SFEBq culture treated with purified laminin (LN) + entactin (EN) and Nodal. n-q, Expression of Mitf (m), CoupTF2 (n), Otx2 (o) and Connexin 43 (p) in the ESC-derived RPE. CoupTF2 + /Chx10 - demarcates the RPE. q, Expression of Chx10 in the E10.5 mouse retina. 2

3 Supplementary Fig. 2 Dynamic morphological changes seen in 3D live imaging with multi-photon optics. a, b, Design of multi-photon microscope for long-term culture. AOM, acousto-optic modulator of laser intensity. RH, relative humidity. PMT, photomultiplier. NDD, 3

4 non-descan detector. c, Relative curvature of the neural retina (or distal epithelium) bending. Note the transition of curving from apically concave to apically convex through a relatively flat period. d, 3D reconstruction views of cell morphology in the RPE, hinge and neural retina (NR) epithelia at Phase 4. Lower panels show the apical views of the apical ends of cells. The apical shape of Phase-4 hinge cells was particularly narrow in the distal-proximal direction. e-g, Expansion of Phase-4 retinal epithelia. e, g, Tangential expansion of the NR (e) and RPE (g; distal portion) tissues during Phase 4. f, Increase of thickness of the NR epithelium (blue). h, Blockade of the NR tissue growth (tangential) by the mitotic inhibitor aphidicolin during Phase 4. i, j, Effects of aphidicolin (5 µm; j) on phospho-histone H3 (ph3) staining (positive control for its efficacy). k, Design of the spinning-disc confocal optic system combined with a multi-gas incubator. 4

5 Supplementary Fig. 3 Regulation of the ROCK-actomyosin system, elasticity and mechanical stress during eye-cup morphogenesis in vitro. a, acetyl-tubulin and pmlc2 staining in the Phase-1 vesicle. b, Temporal profile of the 5

6 inner cavity size (maximal cross-section area; indicated as the yellow area in the inset) during Phase 1. It is considered that the decrease of the cavity size during phase 1 (blue) is dependent on the presence of tension generated by the ROCK-myosin system, since Y treatment (red) substantially reversed it. c, Immunostaining of phalloidin and pmlc2 at Phase-2. d, Quantification of pmlc2 signals along the ESC-derived retinal epithelium at Phase-4. e-f, pmlc immunostaining in the E10.5 mouse retina. The level of pmlc accumulation in the neural retina (NR) was clearly lower than that in the RPE. g, Schematic of elasticity measurement assay of retinal tissues using an AFM-cantilever force probe. The measurement was done with indentation on the apical side of ESC-derived retinal tissues placed on top of a poly-d-lysine-coated plastic culture dish (left. middle). Right, top view of the Phase-4 sample placed on a culture dish for this assay. NR, RPE and NE regions were easily distinguished. h, Relative tissue rigidity of Phase-1 epithelia, which was substantially reduced by the ROCK and actomyosin inhibitors. Similar data were obtained in both proximal and distal regions of Phase-1 vesicles. i, Quantification of the temporal changes of the gap area after 3D multi-photon laser ablation (ratio to the gap area 4 minutes after laser shot)(left). Note that aphidicolin treatment (purple) blocked the gap filling after ablation at Phase 4. Schematic summary for the data in b, h-l is shown on the right. j-l, Laser-created gaps efficiently closed in the Phase-4 RPE (j) or NR (k). In contrast, laser-created gaps remained open in the Phase-1 retinal epithelium (l; also see the red line in i, graph). Rx::GFP (top) and bright-field (bottom) images. m, Chx10 expression in the E9.5 mouse NR primordium. 6

7 Supplementary Fig. 4 Neural retina-vs.-rpe specification in culture of excised vesicles. a-b, Isolated vesicles (without non-retinal neuroectoderm) were cocultured with Wnt3a-expressing L cells that were reaggregated and embedded in undiluted matrigel. c-d, Isolated Phase-1 vesicles (without non-retinal neuroectoderm; NE) were cultured with recombinant Wnt3a protein (200 ng/ml). e-h, k, l, Pax6 (e, g, k) and CoupTF2 (f, h, l) expression in the retinal tissue. e-f, Control SFEBq aggregate. g-h, Treated with the Wnt antagonist IWP2 (4 μm). i-j, No substantial effects of IWP2 on proliferation (ph3 + ; percentage; i) and apoptosis (Tunel + ; j). k-l, Treated with BIO (500 nm). CoupTF2 + /Chx10 - demarcates the RPE. m-q, qpcr analysis of Dkk1 (m), Dkk3 (n), Rx (o), Six3 (p) and N-cad (q) expression in the Rx::GFP + epithelium (each left column) 7

8 and Rx::GFP - NE (each right column) in the day-7 aggregate. r, In situ hybridization showing distally-high expression of Dkk1 in the vesicle at Phase

9 Supplementary Fig. 5 Stratified neural retina tissues formed in long-term 3D culture. 9

10 a, Low magnification view of the sample in Fig. 5a. Cells are partially labeled by co-aggregating Rx::GFP and non-labeled ES cells on day 0. b, Isolated Rx::GFP + neural retina (NR) tissues on day 10. c, Expression of layer-specific markers in the neonatal mouse retina (postnatal day 8). d, Low magnification view of expression of the photoreceptor marker Recoverin in the day-24 ESC-derived retinal tissue. e, Expression of the ganglion cell marker Brn3 (as in the in vivo retina, Brn3 was expressed in a portion of ganglion cells). f, Expression of the Muller glia marker CRALBP in the ESC-derived retina. Top, apical. g-h, The pnrl::dsred2 plasmid (DsRed expression vector under the control of the Nrl promoter) was electroporated apically into ESC-derived retinal tissues on day 16. Nrl is a rod photoreceptor-specific marker. DsRed + cells were detectable on day 18 (h). i, Cryosection of day-20 ESC-derived retinal tissue electroporated with pnrl::dsred and pcag::h2b-venus on day 16. j, Localization of the synaptic marker Bassoon (in synaptic bodies of photoreceptors and in the synapses at the inner plexiform layer) and vglut1 protein (photoreceptor cells). Arrow, vglut - /Bassoon + synapses mimicking the inner plexiform layer; arrowheads, vglut + /Bassoon + ones reminiscent of the outer plexiform layer. k, High-magnification view of aligned cells expressing Calretinin, a marker for amacrine and ganglion cells, in the inner plexiform layer-like zone. l-m, Expression of the cone photoreceptor markers S-opsin (l) and NSE (m) on day 24. It remains to be known whether the relatively low percentage of these cells reflects inefficient differentiation or delayed maturation. n, o, BrdU staining on day 24 in Brn3 + (n) and Chx10 + (o; Pax6 - ) cells that had been incubated with BrdU on day 12 and day 18, respectively. Arrowheads, BrdU-retaining cells. p, q, Expression of Chx10 in day-18 culture with (q) or without (p) DAPT treatment. r, s, DAPT inhibited mitotic marker expression. Expression of Ki67 and ph3 in day-18 ESC-derived retinal tissues with (s) or without (r) DAPT treatment. 10

11 Supplementary Fig. 6 Computer simulation based on the working model. a, Computer-simulated animations based on the three local mechanical conditions (yellow letters). Images corresponding to Phases 1-4 are shown. b, Schematic of the spring model for retinal epithelium. For discretization, vertices are aligned on both the apical and basal surfaces of the epithelium (70 on a half dome of an optic vesicle and 30 on a neighboring non-retinal neuroectoderm (NE) in Phase 1; see the subsequent allotments to RPE, neural retina (NR) and hinge domains of the retinal epithelium at 11

12 Phases 2-4 in the bottom cartoons). Adjacent vertices are connected by apical, basal and transmural elastic springs (length: l a, l b and l t ). This divides the epithelium into small virtual elements (each of which does not represent a cell, but rather conceptually corresponds to a cluster of plural epithelial cells). Tissue expansion in the expanding NR and RPE is represented by the size increase of local virtual elements. Total potential energy is a sum of elastic energy of each spring, volumetric constraint of each element and self-righting potential in the transmural direction (favoring the minimization of the difference between the θ 1 and θ 2 angles on the apical or basal side). The equation of motion (overdamped) for each vertex is as shown (where the parameter η represents the viscosity). r i, position vector of vertex i. c, Invagination of NR failed to occur when Condition 2 (apical constriction initiated at Phase 3) was omitted while the NR was kept rigid. d, e, No substantial effects were seen on the whole cup structure in silico even when strong apical constriction was removed in the late Phase-4 hinge. d, Control (a strongly shortened natural length and a large spring constant were given for the apical hinge spring at Phase 4). e, With substantial relaxation of the apical hinge spring at late Phase 4 (the natural lengths are now 70% of that of the basal spring, respectively, while the spring constants are the same as that of the basal spring). f, g, Computer-simulated eversion of the Phase-4 RPE-hinge after excision at the proximal end of the RPE from the non-retinal neuroectoderm (in this case, simulation was done with relaxation of the Phase-4 apical hinge springs to 40% of the natural length of the basal springs). The plasticity of NR seen in vitro (Fig. 5c) is virtually introduced in silico by pinning the NR tissue near the hinge border. See Supplementary Movie 8c for animation. 12

13 Supplementary Movie Legends Supplementary Movie 1 Evagination of Rx + vesicles from an SFEBq-cultured ESC aggregate. (QuickTime; 2.1 MB) Corresponding to Fig. 1a. Supplementary Movie 2 Eye-cup morphogenesis of ESC-derived retinal tissues in 3D live imaging. (QuickTime; 7.8 MB) Part a, Multi-photon imaging of 3D culture of ESC-derived eye-cup during days Rx::GFP signals were three-dimentionally reconstituted and shown as surface rendering images. Corresponding to Fig. 2b Part b, Multi-photon imaging during days Rx::GFP signals in an optical section were shown together with bright-field images. Corresponding to Fig. 2c Part c, Another example of optic-cup formation in the culture. Part d, 3D reconstruction view of wedge-shaped cells at the Phase-4 hinge. Supplementary Movie 3 Inhibition of invagination by aphidicolin treatment (QuickTime; 1.8 MB) Part a, Invagination of the Phase-4 NR in control culture. Part b, Blocked invagination in culture pretreated with aphidicolin for 5 hours at the end of Phase 3. Supplementary Movie 4 Tissue dynamics responses to 3D-pinpointed cell ablation by multi-photon laser. (QuickTime; 12.8 MB) Part a, Laser ablation in the Phase-4 RPE. Part b, Laser ablation in the Phase-4 NR. Part c, Laser ablation in the Phase-4 hinge epithelium. Part d, Laser ablation in the Phase-4 RPE pretreated with aphidicolin. Part e, Laser ablation in the evaginated Phase-1 epithelium. Supplementary Movie 5 Invagination in the ESC-derived retinal epithelium isolated and cocultured with Wnt-expressing cells. (QuickTime; 2.3 MB) Part a, Control culture of a Phase-1 vesicle without NE. Disc-confocal imaging. Part b, Co-culture with Wnt3a-expressing L cells. Disc-confocal imaging. 13

14 Supplementary Movie 6 Interkinetic nuclear migration in ESC-derived NR tissues. (QuickTime; 4.4 MB) Part a, Multi-photon imaging of neural progenitors in the Phase-4 NR epithelium corresponding to Fig. 5a. Part b, Multi-photon imaging at a higher magnification in the isolated NR epithelium. Nuclear migration was shown by the introduction of H2B-GFP by electroporation of the expression vector. Part c, Expression of Nrl::Venus in the NR tissue during days The Nrl::Venus plasmid was introduced by electroporation on day 16. A dim expression started just before the last division of photoreceptor progenitors and the expression increased soon after it. Supplementary Movie 7 Eversion of the RPE-hinge portion of the Phase-4 cup occurring after excision at the proximal hinge. (QuickTime; 0.5 MB) Corresponding to Fig. 5c. Supplementary Movie 8 Computer-simulated animation of the invagination process. (QuickTime; 2.2 MB) Part a, b The simulated invagination movement is shown in a 2D view (part a) or 3D perspective (part b). Part c The simulated eversion movement of the RPE-hinge after excision at the proximal end of the RPE from the NE. 14