Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture

• Specification of ESC-derived aggregates into eye field-like tissues by Wnt signaling in CDM culture.
• Optic-vesicle structure formation was accompanied by Wnt activation in CDM culture.
• Rax +/Chx10 + retinal progenitors were efficiently generated in CDM culture.

The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM + KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax +/Chx10 + retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro.