Abstract
Purpose :
We previously reported that the T-box transcription factor, Tbx3, is required for normal retina formation. Tbx3 is required downstream of the BMP inhibitor Noggin, maintains eye field neural progenitors in a multipotent state, and with Pax6, drives neural progenitors to form retina. Understanding how Tbx3 expression is regulated during retinal determination will provide insight into the signaling system(s) that direct pluripotent cells to a retinal progenitor fate. In this study, we asked if the BMP and Activin/TGFβ signaling pathways could control tbx3 expression in the eye field.
Methods :
Cycloheximide was used to block protein translation in pluripotent Xenopus laevis animal cap cells. Cap cells and embryos were also treated with chemical inhibitors that repress BMP (Dorsomorphin) and Activin (SB431542) signaling, or both (LDN193189). BMP and Activin/TGFβ signaling were assayed by western blot to detect changes in SMAD phosphorylation. Changes in the expression of tbx3 and other eye field markers were detected using RT-PCR and whole mount in situ hybridization.
Results :
Blocking protein synthesis inhibited the phosphorylation of SMAD1,5,9 (BMP signaling) and induced tbx3 expression in pluripotent animal cap cells, suggesting a link between BMP signaling and tbx3 repression. Inhibition of either BMP (Dorsomorphin) or Activin/TGFβ (SB431542) signaling in the developing embryo, resulted in an expansion of the tbx3 expression domain and the eye field. LDN, which represses both signaling pathways, also expanded the eye field-specific tbx3 expression domain, but also repressed the more anterior cement gland-specific tbx3 expression domain.
Conclusions :
Our results suggest that tbx3 transcription is repressed by BMP and Activin/TGFβ signaling. Noggin blocks BMP and Activin/TGFβ signaling and induces Pax6. Our working hypothesis is that during neural induction, Noggin represses BMP and Activin/TGFβ signaling in the anterior neural plate, resulting in eye field expression of Tbx3, and with Pax6, Tbx3 drives neural progenitors to form retina. Future studies will determine whether downstream targets of these signaling pathways directly regulate tbx3 expression.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.