Abstract
Purpose: :
The purpose of this project is to determine whether retinal precursors can be coerced to reenter the cell cycle when Hes1 replaces Atoh7. The proneural factor Atoh7 is required for cell cycle exit and essential for retinal ganglion cell (RGC) formation. Oppositely, the inhibitor type bHLH factor Hes1 prevents neurogenesis by promoting cell cycle reentry and inhibiting Atoh7 activation. In normal neurogenesis, Hes1 is repeatedly recruited for adding cell numbers. Therefore, we hypothesize that replacing Atoh7 with Hes1 would lead to cell cycle reentry in retinal precursor cells, and that reactivation of Atoh7 after Hes1’s ectopic action would restore RGCs in the retina. We provide here in vivo results for testing these two hypotheses.
Methods: :
An Atoh7(Hes1/Hes1) mouse line carrying a genetic element of loxP-Hes1-IRES-dsRed-loxP in place of Atoh7 coding sequence was generated. Excision of the floxed element would allow an Atoh7-IRES-hrGFP cassette to resume Atoh7’s position. Retinas at different stages were analyzed using proliferation markers to access cell cycle reentry. RNA-sequencing and qRT-PCR were performed to access molecular shifts. The Atoh7(Hes1/Hes1) line was mated to a Cre-ERTM line to generate a Atoh7(Hes1/Hes1);CreERTM line, in which Tamoxifen was administered to reactivate Atoh7 for accessing RGC restoration.
Results: :
The Atoh7(Hes1/Hes1) retinas showed 50% more proliferating cells then the wildtype retinas during early retinogenesis. In consistent with the cell proliferation results, the Atoh7(Hes1/Hes1) retinas also had 50% more total retinal cells then the wildtypes. The high proliferation rate was accompanied by a high apoptotic rate resulting the thinnest adult retina we’ve ever seen. Results of RNA-sequencing and qRT-PCR showed that neurogenic genes were downregulated and cell cycle promoting genes were upregulated. Tamoxifen administration resulted in massive RGC restoration, but some axons appeared to be misrouted forming an ectopic optic disk in each retina examined. It appeared that there was a time shift for RGC production.
Conclusions: :
Additional cell cycles can be coerced in retinal precursors by replacing Atoh7 with Hes1. However, over-proliferated cells cannot survive in such an aberrant retinal environment in which most cells stall in an undifferentiated, or "confused," state. Reactivation of Atoh7 can restore RGC production in vivo with possible time shift into late retinogenesis.
Keywords: retina • retinal development • retina: proximal (bipolar, amacrine, and ganglion cells)