Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Self-organizing optic-cup morphogenesis in three-dimensional culture


Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal–distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Self-formation of an optic-cup-like structure in 3D culture of ES cell aggregates.
Figure 2: Progressive morphogenetic changes of ES-cell-derived retinal epithelium.
Figure 3: Stepwise acquisition of domain-specific epithelial properties.
Figure 4: Self-patterning into neural retina and RPE via interactions with neuroectodermal epithelium.
Figure 5: Generation of stratified neural retina tissues from ES-cell-derived invaginated epithelia.


  1. Spemann, H. Ueber korrelationen in der entwicklung des auges. Verh. Anat. Ges. 15, 61–79 (1901)

    Google Scholar 

  2. Lewis, W. H. Experimental studies on the development of the eye in Amphibia I. On the origin of the lens in Rana palustrius . Am. J. Anat. 3, 505–536 (1904)

    Article  Google Scholar 

  3. Nakagawa, S., Takada, S., Takada, R. & Takeichi, M. Identification of the laminar-inducing factor: Wnt-signal from the anterior rim induces correct laminar formation of the neural retina in vitro . Dev. Biol. 260, 414–425 (2003)

    Article  CAS  Google Scholar 

  4. Martinez-Morales, J. R., Rodrigo, I. & Bovolenta, P. Eye development: a view from the retina pigmented epithelium. Bioessays 26, 766–777 (2004)

    Article  CAS  Google Scholar 

  5. Mu, X. & Klein, W. H. A gene regulatory hierarchy for retinal ganglion cell specification and differentiation. Semin. Cell Dev. Biol. 15, 115–123 (2004)

    Article  CAS  Google Scholar 

  6. Wilson, S. W. & Houart, C. Early steps in the development of the forebrain. Dev. Cell 6, 167–181 (2004)

    Article  CAS  Google Scholar 

  7. Rembold, M., Loosli, F., Adams, R. J. & Wittbrodt, J. Individual cell migration serves as the driving force for optic vesicle evagination. Science 313, 1130–1134 (2006)

    Article  ADS  CAS  Google Scholar 

  8. Cayouette, M., Poggi, L. & Harris, W. A. Lineage in the vertebrate retina. Trends Neurosci. 29, 563–570 (2006)

    Article  CAS  Google Scholar 

  9. Adler, R. & Canto-Soler, M. V. Molecular mechanisms of optic vesicle development: complexities, ambiguities and controversies. Dev. Biol. 305, 1–13 (2007)

    Article  CAS  Google Scholar 

  10. Martinez-Morales, J. R. & Wittbrodt, J. Shaping the vertebrate eye. Curr. Opin. Genet. Dev. 19, 511–517 (2009)

    Article  CAS  Google Scholar 

  11. Picker, A. et al. Dynamic coupling of pattern formation and morphogenesis in the developing vertebrate retina. PLoS Biol. 7, e1000214 (2009)

    Article  Google Scholar 

  12. Fuhrmann, S. Eye morphogenesis and patterning of the optic vesicle. Curr. Top. Dev. Biol. 93, 61–84 (2010)

    Article  Google Scholar 

  13. Livesey, F. J. & Cepko, C. L. Vertebrate neural cell-fate determination: lessons from the retina. Nature Rev. Neurosci. 2, 109–118 (2001)

    Article  CAS  Google Scholar 

  14. Bailey, T. J. et al. Regulation of vertebrate eye development by Rx genes. Int. J. Dev. Biol. 48, 761–770 (2004)

    Article  CAS  Google Scholar 

  15. Lopashov, G. Developmental Mechanism of Vertebrate Eye Rudiments (Pergammon, 1963)

    Google Scholar 

  16. Hyer, J., Mima, T. & Mikawa, T. FGF1 patterns the optic vesicle by directing the placement of the neural retina domain. Development 125, 869–877 (1998)

    CAS  PubMed  Google Scholar 

  17. Hyer, J., Kuhlman, J., Afif, E. & Mikawa, T. Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation. Dev. Biol. 259, 351–363 (2003)

    Article  CAS  Google Scholar 

  18. Smith, A. N., Miller, L. A., Radice, G., Ashery-Padan, R. & Lang, R. A. Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis. Development 136, 2977–2985 (2009)

    Article  CAS  Google Scholar 

  19. Wataya, T. et al. Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation. Proc. Natl Acad. Sci. USA 105, 11796–11801 (2008)

    Article  ADS  CAS  Google Scholar 

  20. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008)

    Article  CAS  Google Scholar 

  21. Au, E. & Fishell, G. Cortex shatters the glass ceiling. Cell Stem Cell 3, 472–474 (2008)

    Article  CAS  Google Scholar 

  22. Ikeda, H. et al. Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc. Natl Acad. Sci. USA 102, 11331–11336 (2005)

    Article  ADS  CAS  Google Scholar 

  23. Fujiwara, H. et al. Regulation of mesodermal differentiation of mouse embryonic stem cells by basement membranes. J. Biol. Chem. 282, 29701–29711 (2007)

    Article  CAS  Google Scholar 

  24. Lagutin, O. et al. Six3 promotes the formation of ectopic optic vesicle-like structures in mouse embryos. Dev. Dyn. 221, 342–349 (2001)

    Article  CAS  Google Scholar 

  25. Tang, K. et al. COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis. Development 137, 725–734 (2010)

    Article  CAS  Google Scholar 

  26. Hilfer, S. R. & Yang, J.-J. W. Accumulation of CPC-precipitable material at apical cell surfaces during formation of the optic cup. Anat. Rec. 197, 423–433 (1980)

    Article  CAS  Google Scholar 

  27. Sawyer, J. M. et al. Apical constriction: a cell shape change that can drive morphogenesis. Dev. Biol. 341, 5–19 (2010)

    Article  CAS  Google Scholar 

  28. Kinoshita, N., Sasai, N., Misaki, K. & Yonemura, S. Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation. Mol. Biol. Cell 19, 2289–2299 (2008)

    Article  CAS  Google Scholar 

  29. Agius, E. et al. Converse control of oligodendrocyte and astrocyte lineage development by Sonic hedgehog in the chick spinal cord. Dev. Biol. 270, 308–321 (2004)

    Article  CAS  Google Scholar 

  30. Amano, M. et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271, 20246–20249 (1996)

    Article  CAS  Google Scholar 

  31. Schoenwolf, G. C. & Smith, J. L. Mechanisms of neurulation: traditional viewpoint and recent advances. Development 109, 243–270 (1990)

    CAS  PubMed  Google Scholar 

  32. Haigo, S. L., Hildebrand, J. D., Harland, R. M. & Wallingford, J. B. Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr. Biol. 13, 2125–2137 (2003)

    Article  CAS  Google Scholar 

  33. Hildebrand, J. D. Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network. J. Cell Sci. 118, 5191–5203 (2005)

    Article  CAS  Google Scholar 

  34. Nishimura, T. & Takeichi, M. Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling. Development 135, 1493–1502 (2008)

    Article  CAS  Google Scholar 

  35. Rico, F. et al. Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips. Phys. Rev. E 72, 021914 (2005)

    Article  ADS  Google Scholar 

  36. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nature Cell Biol. 10, 429–436 (2008)

    Article  CAS  Google Scholar 

  37. Mascaro, A. L., Sacconi, L. & Pavone, F. S. Multi-photon nanosurgery in live brain. Front. Neuroenergetics 2, (2010)

  38. Fuhrmann, S. Wnt signaling in eye organogenesis. Organogenesis 4, 60–67 (2008)

    Article  Google Scholar 

  39. Westenskow, P., Piccolo, S. & Fuhrmann, S. β-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development 136, 2505–2510 (2009)

    Article  CAS  Google Scholar 

  40. Liu, W., Lagutin, O., Swindell, E., Jamrich, M. & Oliver, G. Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate. J. Clin. Invest. 120, 3568–3577 (2010)

    Article  CAS  Google Scholar 

  41. Fujimura, N., Taketo, M. M., Mori, M., Korinek, V. & Kozmik, Z. Spatial and temporal regulation of Wnt/β-catenin signaling is essential for development of the retinal pigment epithelium. Dev. Biol. 334, 31–45 (2009)

    Article  CAS  Google Scholar 

  42. Yang, X. J. Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis. Semin. Cell Dev. Biol. 15, 91–103 (2004)

    Article  CAS  Google Scholar 

  43. Diep, D. B., Hoen, N., Backman, M., Machon, O. & Krauss, S. Characterisation of the Wnt antagonists and their response to conditionally activated Wnt signalling in the developing mouse forebrain. Brain Res. Dev. Brain Res. 153, 261–270 (2004)

    Article  CAS  Google Scholar 

  44. Norden, C., Young, S., Link, B. A. & Harris, W. A. Actomyosin is the main driver of interkinetic nuclear migration in the retina. Cell 138, 1195–1208 (2009)

    Article  CAS  Google Scholar 

  45. Pinzón-Duarte, G., Kohler, K., Arango-Gonzalez, B. & Guenther, E. Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture. Vision Res. 40, 3455–3465 (2000)

    Article  Google Scholar 

  46. Matsuda, T. & Cepko, C. L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl Acad. Sci. USA 104, 1027–1032 (2007)

    Article  ADS  CAS  Google Scholar 

  47. Stella, S. L., Jr, Li, S., Sabatini, A., Vila, A. & Brecha, N. C. Comparison of the ontogeny of the vesicular glutamate transporter 3 (VGLUT3) with VGLUT1 and VGLUT2 in the rat retina. Brain Res. 1215, 20–29 (2008)

    Article  CAS  Google Scholar 

  48. Fuhrmann, S., Levine, E. M. & Reh, T. A. Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127, 4599–4609 (2000)

    CAS  PubMed  Google Scholar 

  49. Cavodeassi, F. et al. Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/β-catenin pathway. Neuron 47, 43–56 (2005)

    Article  CAS  Google Scholar 

  50. Seiler, M. J. et al. Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets. Eur. J. Neurosci. 31, 508–520 (2010)

    Article  CAS  Google Scholar 

  51. Honda, H., Tanemura, M. & Nagai, T. A three-dimensional vertex dynamics cell model of space-filling polyhedra simulating cell behaviour in a cell aggregate. J. Theor. Biol. 226, 439–453 (2004)

    Article  Google Scholar 

  52. Nagai, T. & Honda, H. Computer simulation of wound closure in epithelial tissues: cell-basal-lamina adhesion. Phys. Rev. E 80, 061903 (2009)

    Article  ADS  Google Scholar 

  53. Inoue, Y. & Adachi, T. Coarse-grained Brownian ratchet model of membrane protrusion on cellular scale. Biomech. Model. Mechanobiol. 10.1007/s10237-010-0250-6 (19 August 2010)

Download references


We are grateful to S. Nakanishi, S. Yonemura, R. Ladher, K. Muguruma, H. Inomata and M. Ohgushi for comments and to members of the Sasai laboratory for discussion. We also thank Olympus, particularly Y. Saito, M. Suzuki and Y. Imai, for their help and discussion regarding the design, assembly and optimized utility of the incubator-combined multi-photon and confocal optic systems, and T. Sugitate and N. Saito at JPK Instruments for technical advice on the AFM assay. This work was supported by grants-in-aid from MEXT (Y.S., M.E., T.A.), the Knowledge Cluster Initiative at Kobe and the S-Innovation Project (Y.S., K.S.), and the Leading Project for Realization of Regenerative Medicine (Y.S).

Author information

Authors and Affiliations



M.E. and Y.S. designed the project and wrote the manuscript. M.E., N.T., M.K. and E.S performed experiments. K.S. provided critical technical information on matrix experiments. H.I., S.O. and T.A. performed computer simulation by discussing details with M.E. and Y.S.

Corresponding authors

Correspondence to Mototsugu Eiraku or Yoshiki Sasai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-6 with legends and Legends for Supplementary Movies 1-8. (PDF 1055 kb)

Supplementary Movie 1

This movie shows evagination of Rx+ vesicles from an SFEBq-cultured ESC aggregate. (MOV 2195 kb)

Supplementary Movie 2

This movie shows eye-cup morphogenesis of ESC-derived retinal tissues in 3D live imaging (MOV 7884 kb)

Supplementary Movie 3

This movie shows inhibition of invagination by aphidicolin treatment. (MOV 774 kb)

Supplementary Movie 4

This movie shows tissue dynamics responses to 3D-pinpointed cell ablation by multi photon laser. (MOV 14187 kb)

Supplementary Movie 5

This movie shows invagination in the ESC-derived retinal epithelium isolated and cocultured with Wnt-expressing cells. (MOV 2382 kb)

Supplementary Movie 6

This movie shows interkinetic nuclear migration in ESC-derived NR tissues. (MOV 6564 kb)

Supplementary Movie 7

This movie shows eversion of the RPE-hinge portion of the Phase-4 cup occurring after excision at the proximal hinge. (MOV 509 kb)

Supplementary Movie 8

This movie shows computer-simulated animation of the invagination process. (MOV 2129 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Eiraku, M., Takata, N., Ishibashi, H. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing