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

Abstract

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.

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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.

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Acknowledgements

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).

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Authors and Affiliations

Authors

Contributions

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.

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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)

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Eiraku, M., Takata, N., Ishibashi, H. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011). https://doi.org/10.1038/nature09941

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