Abstract
Purpose: :
Following stimulation of retinal stem cell proliferation, controlling the cell fate choices of progenitor cells remains a key obstacle to developing regenerative therapeutics for retinal disease. Activation of the Wnt pathway stimulates adult retinal stem cell proliferation in mammals and zebrafish. However, subsequent roles for Wnt signaling in progenitor cell differentiation are not well defined. Here, we examined the role of the Wnt pathway in regulating proliferation and differentiation of retinal stem cells and their progenitors, respectively, following selective retinal bipolar cell loss in late stage zebrafish larvae.
Methods: :
We use a prodrug converting enzyme system for inducible cell ablation as a means of studying the regeneration of specific retinal cell subtypes in zebrafish. Transgenic expression of nitroreductase (NTR) and a fluorescent reporter renders NTR-expressing cells susceptible to prodrug induced death. After the prodrug is removed, the lost cells are replaced. The fluorescent reporter allows the regenerative process to be followed in late-stage larvae using high-resolution time lapse confocal microscopy. This approach was combined with genetic and chemical modulations of Wnt effectors to test for effects on the kinetics of retinal bipolar cell regeneration. In particular, to evaluate age-dependent effects of Wnt signaling on stem cell proliferation and progenitor differentiation.
Results: :
Retinal stem cell responses to Wnt signaling were fundamentally different in late-stage larvae compared to adults. Moreover, Wnt signaling played a direct role in promoting bipolar cell fates during regeneration. Importantly, reciprocal modulations of the Wnt pathway that first increased stem cell proliferation and then promoted bipolar cell differentiation resulted in enhanced retinal bipolar cell regeneration.
Conclusions: :
Our targeted cell ablation approach provides a means to investigate cell-type and age-dependent specificities of retinal regeneration processes in zebrafish. This study revealed surprising age-dependent differences in how Wnt signaling regulates retinal regeneration and direct effects on cellular differentiation. The results suggest that congenital and age-related degenerative retinal diseases may require fundamentally different regenerative treatment strategies.
Keywords: regeneration • bipolar cells • Muller cells