Abstract
Purpose: :
FoxO3 is a forkhead transcription factor that regulates genes in control of cell cycle and apoptotic pathways. The Xenopus laevis FoxO3 (xlFoxO3) homologue is highly conserved compared to mouse and human proteins. We hypothesized that xlFoxO3 has a role in the regulation of cell cycle in RPCs during vertebrate eye development
Methods: :
We confirmed xlFoxO3 is expressed in retinal progenitor cells (RPCs) of developing X. laevis eyes. We produced a phosphorylation mutant (T30A) at a residue shown to dictate nuclear localization of the FoxO3 protein. Wt or T30A xlFoxO3 was misexpressed in the anterior neural tissue by RNA injection into one dorsal blastomere of four-cell stage embryos; GFP RNA was co-injected to confirm properly targeted expression.
Results: :
Targeted expression of wt or T30A xlFoxO3 results in embryos with delayed retinal pigmentation and small eyes. The optic cups of FoxO3 injected embryos appear morphologically immature compared to optic cups in uninjected control embryos. Exogenous T30A xlFoxO3 increases the frequency and severity of embryos exhibiting the small eye phenotype compared to wt xlFoxO3. Exogenous xlFoxO3 does not affect the presence of amacrine, ganglion, horizontal or photoreceptor cells in the mature retina, evidenced by positive Islet1 and RetP1 staining. During early retinal histogenesis, wt and T30A xlFoxO3 injected retinae have reduced expression of the neural precursor gene, N-Myc and the cell cycle markers, CyclinD1 and p27. We analyzed the gene expression profile of differentiating RPCs using markers expressed in RPCs at progressive stages of differentiation. Otx2 expression is reduced in the optic cups when FoxO3 (wt or T30A) is overexpresed. Pax6 expression is reduced in FoxO3 injected retinae during early retinohistogenesis. Markers of differentiating retinal progenitors NeuroD, Notch are reduced in retinae injected with FoxO3 (wt or T30A). Expression of Otx5b, a marker of differentiating photoreceptor cells, is reduced in injected retinae.
Conclusions: :
The data demonstrates that xlFoxO3 plays a critical role in vertebrate eye development: xlFoxO3 affects expression of cell cycle genes and genes involved in differentiation of retinal progenitor cells, thereby regulating the development of RPCs that contribute to the neural retina.
Keywords: development • gene/expression • transcription factors