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Sheik Pran Babu Sardar Pasha, Federico Calegari, Mike.O Karl; Ccnd1-Cdk4 stimulates Müller glia cell cycle re-entry in the mouse retina. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Adult mammalian retinas have a limited potential to regenerate retinal neurons. Major restrictions of retinal regeneration are a low number of Müller glia (MG) re-entering the cell cycle and insufficient progeny generation. Here, we investigated animal age-dependence of the MG proliferative response and explored ways of its stimulation upon retina damage.
Mouse retinas were damaged by neurotoxin NMDA at various ages, and mitogens were applied to stimulate MG proliferation. EdU was administered to label proliferating cells. Doxycycline was used to activate the cell cycle regulator Ccnd1 and Cdk4 (4D) complex in hGFAP-Cre::Rosa-rtTA::tetO-4D-RFP transgenic mouse retina. We analyzed MG cell proliferation (EdU+ Sox9+) over 7 days post injury (dpi) in retinal wholemount or sections using immunostaining and confocal microscopy.
Müller glia proliferation is a very rare event in the NMDA damaged mouse retina. Upon additional HB-EGF stimulation, we observed the highest MG proliferative response after 7 dpi in juvenile mice (38 ±2 SEM EdU+ Sox9+ cells per mm; N=4); with a 3.5-fold increase compared to EGF and EGF plus TGF-β inhibitor. Notably, the MG proliferation response decreased significantly with increasing animal age (postnatal day (P); P10: 38 ±2 SEM versus P12: 2.53 ±0.1 SEM Sox9+EdU+ cells; N=4; ***p<0.001). Conditionally induced 4D transgene expression significantly stimulated proliferation and expansion of MG derived cell progeny (1025 ±64 SEM RFP+ EdU+ cells per mm; N=4; ***p<0.001) in juvenile damaged retina. MG proliferation was confirmed by analyzing expression of various proliferation markers. 4D transgene expression leads to about a 5-fold increase in Ki67 cells per mm (364 ±8 SEM; N=4,**p<0.01) compared to HB-EGF (67 ±3 SEM; N=4) treated retina.
Higher numbers of MG proliferation could be stimulated in juvenile retina compared to any previous report in the literature. Our preliminary data suggests the possibility of MG cell cycle re-entry and de-differentiation depend on animal age and appropriate mitogen stimulation. Notably, increased expansion of MG progeny can be induced genetically by overexpression of the 4D complex upon retina damage. Thus, it will be of great interest to find out the fate of MG-derived progeny, and the mechanisms that regulate and restrict Müller glia proliferation and stem cell competence.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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