Otx2 directly regulates tbx3 expression during early eye formation

Eye field specification requires precise spatiotemporal signaling. Among these signals, the morphogen Noggin blocks both BMP and Activin signaling pathways to generate retinal tissue from pluripotent frog animal cap cells. We previously found that Pax6 and Tbx3 are sufficient to replace Noggin in generating retinal tissue, but it was unclear which signaling pathway affected tbx3 expression. We had also found that Noggin, while activating all other eye field transcription factors (EFTFs), repressed tbx3 expression in animal cap cells, and that Otx2, a transcription factor essential for anterior neural plate induction, is expressed prior to tbx3 and can overcome Noggin-mediated repression to induce tbx3 in vitro. Since we have also found that Tbx3 is required for eye formation, we sought to determine how it is regulated. Here, we show that in vivo tbx3 expression is affected by both BMP and Activin signaling, and that the BMP/Activin chemical inhibitor LDN-193189 preferentially expands tbx3 expression in the eye field while repressing the cement gland, the anterior-most structure in the frog embryo. We further show that otx2, tbx3 and rax expression are all expanded in the eye field following LDN-193189 treatment. To determine how tbx3 is regulated in the eye, we identify evolutionarily conserved regions of the tbx3 gene that drive GFP expression in the eyes of transgenic tadpoles. We observed direct binding by myc-tagged Otx2 to activate tbx3 and one region that myc-tagged Otx2 directly binds. Together, these findings close the regulatory loop, demonstrating that tbx3 expression and early eye formation are directly regulated by Otx2-mediated activation.