Deletion of the imprinted gene Plagl1 activates the proliferative and regenerative capacity of mammalian Müller glia
Description
To determine whether Plagl1 has an essential role in the postnatal retina, we used Plagl1+/-pat mice carrying a silenced maternal allele and a null mutation in the expressed paternal allele; these mice were previously confirmed to lack Plagl1 expression in various tissues, including the embryonic retina. Dysmorphologies were observed in Plagl1+/-pat retinas that phenocopied observations made in various mutant mice with underlying Müller glia defects. We thus examined Plagl1+/-pat retinas for signs of reactive gliosis, a stress-induced Müller glia response associated with cellular hypertrophy and increased expression of ERK signaling and reduced expression of Glul expression. In Figure 3 Western blots, we show the loss of Plagl1 induces Müller glia gliosis. (E) Western blotting for pERK in P14 wild-type and Plagl1+/-pat retinas showing increase in pERK protein levels. N=6 for both wild-type and Plagl1+/-pat retinas. (F) Western blotting for Glul in P14 wild-type and Plagl1+/-pat retinas showing decrease of Glul protein levels. N=6 for both wild-type and Plagl1+/-pat retinas.
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Retinas were collected from adult and postnatal pups at the indicated stages, lysed in RIPA buffer with protease (1x protease inhibitor complete, 1 mM PMSF) and phosphatase (50 mM NaF, 1 mM NaOV) inhibitors, and 10µg of lysate was run on 10% SDS-PAGE gels for Western blot analysis as described previously (Ma et al., 2007). Primary antibodies included pERK (Map kinase p44/42 phospho-Thr202/Tyr204; Cell Signaling #4370), ERK (Map kinase p44/42; Cell Signaling #9102), Glul (Abcam, #ab73593, 1/10.000) and Actin (Abcam; #ab8227, 1/10.000). Densitometries were calculated using ImageJ. The average values of normalized expression levels were plotted from N=6 per genotype.