Abstract
Purpose: :
Neuronal regeneration in the retina of a few non-mammalian animals is complete. In some species Muller glia cells de-differentiate into progenitor-like cells and regenerate all types of retinal neurons. Recent studies suggest that in rodents a very restricted regenerative program can be reactivated. Out ultimate goal is to indentify and overcome the barriers of regeneration in mammals.
Methods: :
Mouse retinas were isolated at postnatal (P) ages P6-30 and cultured as explants. We applied various mitogens to stimulate proliferation. We monitored proliferation by BrdU incorporation. We identified cells based on various fluorescent genetic reporter mice (Muller glia: GFAP-Cre::R-YFP) as well as by immunostaining for SOX9 or SOX2 (Muller glia or progenitor) and ASCL1 (retinal progenitor).
Results: :
Retinal development in mice is finished in the central retina at P5 and at P10-11 no progenitors can be found in the entire retina any more. Retinal explantation leads to ongoing neuronal cell death in vitro. Mitogens stimulated Muller glia cell cycle re-entry in the central retina at ages P6 to P14 at high numbers in vitro. Proliferation started after 3 days in vitro (DIV) and further increased over time. Proliferation was stimulated by growth factors: EGF > FGF = SHH = WNT3a. In P12 retinas at DIV6 with EGF we observed 31 +/-11 SEM (100um retina length; n=5) BrdU positive cells out of which 53% +/-9 SEM were SOX9+ BrdU+ double-positive. The total number of Muller glia re-entering the cell cycle decreased with animal age. No significant proliferation was observed in >P16 retinas. Moreover, we observed re-expression of retinal progenitor genes in proliferating Muller glia including PAX6 and ASCL1, which was as well animal age-dependent.
Conclusions: :
We previously showed that in the adult mouse retina in vivo a very limited number of Muller glia may regenerate an even smaller number of neurons. We now found that right after retinogenesis is complete the still young Muller glia de-differentiate and re-enter the cell cycle at high numbers in vitro and that this plasticity gets restricted with age. Our data suggests that in postmitotic Muller glia mechanisms exist that establish roadblocks to the regenerative program. In ongoing studies we are exploring these mechanisms and test whether young glia regenerate more types and numbers of neurons compared to adult glia.
Keywords: regeneration • retinal glia • retinal development