Optic cup derived from mouse ES cells. GFP shows expression of Rx. Image: RIKEN |
Developmental
processes are increasingly well-characterized at the molecular and cell
biological levels, but how more complex tissues and organs involving
the coordinated action of multiple cell types in three dimensions is
achieved remains something of a black box. One question of particular
interest and importance is whether signaling interactions between
neighboring tissues are essential to guiding organogenesis, or whether
these can arise autonomously from developmental routines inherent to a
given primordial tissue. Finding answers to these questions will be
critical both to a better understanding of embryonic phenomena and to
the ability to control the differentiation of cell populations into
desired configurations.
A breakthrough new report by Mototsugu Eiraku, deputy leader of the Four-dimensional Tissue Analysis Unit and colleagues in the Laboratory for Neurogenesis and Organogenesis (Group Director, Yoshiki Sasai),
as well as the RIKEN VCAD Program, and Kyoto and Osaka Universities,
describes how mouse embryonic stem cells (ESCs) are able to
differentiate and assemble into an optic cup, capable of giving rise to a
tissue exhibiting the stratified structure characteristic of the retina
in vivo. Published in Nature,
the study used a cutting-edge three-dimensional tissue culture system
not only to demonstrate this self-organizing capacity of pluripotent
stem cells, but the underlying cell dynamics as well.
The
mechanistic basis for the formation of the optic cup, with its complex
two-walled structure, has been a longstanding question in embryology.
The retina, with its origins in the lateral midbrain, is part of the
central nervous system. Its development begins with the formation of the
optic vesicle, a pocket of epithelium that deepens and pinches to form
the optic cup, which develops a double layer of cells, with pigment
epithelium on the outer, and neural retina on the inner wall. It has
generally been thought that this transformation is triggered by chemical
and physical influences from other tissues, such as lens or cornea, but
some, including the father of experimental embryology, Hans Spemann,
have suggested that perhaps external induction or force is not
necessary.
To
resolve this question, Eiraku et al. built on a series of techniques
and findings emerging from the use of the SFEBq (serum-free culture of
embryoid body–like aggregates) ES cell culture system developed by the
Sasai lab, which had previously been used to differentiate these
pluripotent stem cells into a wide range of neuronal cell types,
including, recently, structurally organized cerebral cortical neurons.
By adding extracellular matrix proteins to the SFEBq medium, the group
was able to epithelially-organized retinal precursors at high
efficiencies by day 7 of culture. One day later, optic vesicle-like
structure began to form, followed by bi-layered optic cup-like
structures by day 10. The pigmented and neuronal character of the outer
and inner layers of cells in these spontaneously formed tissues were
confirmed by gene expression, indicating that optic cup development had
been recapitulated in vitro, and importantly, in the absence of any
external signaling sources, such as lens, demonstrating the capacity for
self-organization.
They
next used multi-photon microscopy to explore the mechanisms behind this
process of self-assembly in 3D. They found that after the ES
cell-derived retinal precursors differentiated into pigmented epithelial
and neuronal layers, the tissue underwent a four step morphological
rearrangement on its way to assuming the optic cup structure. When they
examined cytoskeletal behaviors in this process, they noted that myosin
activity dropped in the region of the epithelium that bend inward to
form the cup, giving the flexibility needed to form a pocket driven by
expansion of the epithelium through cell division.
Computer
simulation of the mechanics behind this revealed that three principal
forces can explain the optic cup-forming event. First, the a region of
the epithelium must lose rigidity, allowing it to buckle inward, after
which cells at the hinge points (defined by the border between
presumptive pigment epithelium and neuronal regions) must undergo apical
constriction, giving them a wedge-like shape. Once these conditions are
met, expansion of the tissue surface by cell division results further
involution of the cup, all of which are very much in line with the
experimental findings.
As
a final test of the in vitro structure’s ability to mirror its
embryonic counterpart, Eiraku excised the neuronal layer from the ES
cell-derived optic cup and allowed it to develop in 3D cell culture
under conditions optimized for spurring neuronal maturation. He found
that the retinal neurons underwent active mitosis and ultimately
organized into a six-layer stratified and synapse-forming neuronal
structure closely resembling that of the post-natal retina.
“What
we’ve been able to do in this study is resolve a nearly century-old
problem in embryology, by showing that retinal precursors have the
inherent ability to give rise to the complex structure of the optic
cup,” says Sasai. “It’s exciting to think that we are now well on the
way to becoming able to generate not only differentiated cell types, but
organized tissues from ES and iPS cells, which may open new avenues
toward applications in regenerative medicine.” Potential applications
include regenerative medicine approaches to the treatment of retinal
degenerative disorders, such as retinitis pigmentosa.