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The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System
Published on: August 28, 2010
Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic
Eriko Sakakura1, Mototsugu Eiraku1, Nozomu Takata2
1Laboratory for in vitro Histogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Abstract:
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.
