Self-organizing optic-cup morphogenesis in three-

dimensional cultureMototsugu Eiraku, Nozomu Takata, Hiroki Ishibashi,

Masako Kawada, Eriko Sakakura, Satoru Okuda, Kiyotoshi Sekiguchi, Taiji Adachi & Yoshiki Sasai

Nature 472, 51–56, 2011

Intro

• In vivo eye development in mouse• Self-organizing optic cup morphogenesis• Self-patterning of specific neural retinal

domains• Self-directed stratification of neural retina

tissue• Conclusions• Discussion

In Vivo Eye Development (Mouse)

E8.5 E9.5 E10.5E11.5

• Neuroectoderm – optic vesicle (optic cup, neural retina, retinal pigment epithelium), optic nerve

• Surface ectoderm - Lens

Molecular mechanisms of optic development (Adler, Canto-Soler 2007)

Molecular mechanisms of optic development (Adler, Canto-Soler 2007)

Optic-cup self-formation in 3D ES cell culture

• What does self-organizing morphogenesis of the optic cup look like?

• What factors may or may not play a role? (i.e. lens/surface ectoderm, neuroepithelium)

• What are the characteristics of the differentiated cells?

• In vitro vs. in vivo (self-assembly, patterning, morphogenesis)

What does self-organizing morphogenesis of the optic cup look like?

• ES cell aggregate Rx+ GFP+ neuroepithelium – Activin, laminin, entactin, Nodal

• See what happens…

Evagination of optic vesicles

Evagination of optic vesicles

Rx-GFP+ vesicles are Pax+ Sox1-, consistent with retinal marker expression in mouse embryos

Flattening and invagination of optic vesicle

Flattening and invagination of optic vesicle

• Differentiation of the RPE and NR• RPE –Pax6, Mitf expression,

observable pigment, ↓ Rx expression

• NR – Rx, Chx10 expression• Apical (aPKC) – Basal (Laminin)

polarity, same as in vivo

Overview of self-organizing morphogenesis

RPE, NR, and hinge cells• Invagination occurs as cells

proliferate, differentiate

• Distinct morphologies, as in vivo

• Distinct actomyosin activation, RPE vs. NR

• Distinct gene-expression

RPE, NR, and hinge cells

Self-Patterning into neural retina and RPE domains

• Vesicles with neighboring neuroectodermal epithelium invaginated and formed optic cup

• (-)NE vesicles – no RPE differentiation, NR develops

• Wnt3 expressing cells significantly rescue RPE differentiation

• RPE patterning is dependent on tissue interactions

• NR develops autonomously

Self-Patterning into neural retina and RPE domains

Self-directed stratification of neural retina tissue

• Excise prolonged culture

Self-directed stratification of neural retina tissue

Self-directed stratification of neural retina tissue

ONL – outer nuclear layer

INL – inner nuclear layer

GCL – ganglion cell layer

PR – photoreceptors

BP – bipolar cells

Self-directed stratification of neural retina tissue

ES neural retina tissue

Conclusions• Development of the optic cup does not depend on forces (chemical

or physical) of external structures (lens, surface ectoderm)

• Self-formed retinal epithelium domains have distinct morphological, mechanical, and gene-expressing properties

• RPE differentiation is dependent upon induction factors from neuroepithelium, NR differentiation is autonomous

• ES neural retina tissue self-forms in a manner consistent with the spatiotemporal order seen in vivo

• Assembly, patterning, and morphogenesis of the retinal optic cup is self-directed and self-organized.

Discussion

• What does this mean? How can it be useful?

• In vitro vs. in vivo, can organoids fully resemble functional organs?

• Future research? Experimental approach?