The eye field is first specified in the anterior neural plate as the eye primordium, which eventually forms the visible optic cup via evagination of the diencephalic wall.
19 This region can be identified by specific expression of EFTFs, such as Pax6, Rx, Six3, and Lhx2.
19 Previous studies showed that the anterior fate of the neuroectoderm is greatly enhanced upon inhibition of canonical Wnt signaling.
29 Thus, we first tested whether Wnt signal inhibition facilitates anteriorization of the neuroectoderm as well as eye field specification. To this end, we employed XAV, a potent tankyrase inhibitor, to inhibit canonical Wnt signaling.
30 We treated EBs with 1 μM XAV for 4 days in the presence of small molecule inhibitors of BMP and TGFβ. Quantitative gene expression analysis of neuroectoderm markers and the anteroposterior axis revealed that XAV treatment significantly upregulated the expression of a forebrain marker,
BF1 (also known as
FOXG1), whereas such treatment downregulated expression of mid- and hindbrain markers,
EN1 and
GBX2, compared to levels in the DM and SB-treated control group (
Fig. 1A). The expression level of
SOX1, a pan-neural marker gene, was not significantly different in XAV-treated cells compared to in a control (
Fig. 1A). We next examined whether Wnt inhibition promotes the expression of EFTFs. As shown in
Figure 1B, treatment of EB-stage cells with 0.5 to 1 μM XAV for 4 days significantly enhanced the expression of EFTFs. XAV treatment with 1.0 μM did not significantly increase
PAX6 expression compared to the control condition (DM and SB only), while 0.5 μM of XAV did increase
PAX6 expression. However, the fold-increase in
PAX6 by XAV treatment was marginal at both concentrations (1.56-fold at 0.5 μM and 1.13-fold at 1.0 μM), and upregulation of
RX and
SIX3 by XAV treatment was robust at 1.0 μM and displayed a clear dosage-dependency (4.11- and 3.67-fold at 0.5 μM and 18.46- and 12.95-fold at 1.0 μM). Therefore, we used 1.0 μM XAV for retinal induction.