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. 2018 Feb 28;38(9):2246–2261. doi: 10.1523/JNEUROSCI.2126-17.2018

Figure 8.

Figure 8.

Ascl1 and Lin28a expression enhances survival of proliferating MG and regenerate multiple neuron types in the injured retina. A, Experimental timeline. B, Lin28a expression when combined with Ascl1 expression enhances survival of proliferating MG in the injured retina compared with Ascl1 expression alone. Sox9 and BrdU immunofluorescence on retinal sections were used to quantify MG proliferation; n = 5 different experiments. Error bars are SD. C, Representative images of lineage traced BrdU+ cells in the injured retina suggests that most proliferating cells in the Ascl1- and Lin28a-expressing retina are Sox9+ MG (arrows). Arrowheads indicate 4C4+ microglia. Scale bar, 50 μm. D, Quantification of data shown in C; n = 5 different experiments. Error bars are SD. E, Lineage tracing experiments were performed as indicated in A and BrdU+ cells (red) with cell-type-specific markers (green) of photoreceptors (Crx), bipolar (Chx10), amacrine (AP2), and ganglion cells (RBPMS) are indicated by arrows. F, Dox-dependent expression of GFP in eyes that received intravitreal injection of a mixture of AAV ShH10-tetCMV:GFP, AAV ShH10-tetCMV:Ascl1, and AAV ShH10-tetCMV:Lin28a. G, Timeline and quantification of lineage trace experiment in hsp70:ascl1a;hsp70:lin28a fish treated with heat shock for 4 or 15 d when using EdU labeling at 4 d post-heat shock. Retinal cell-type antibodies were used to quantify the number of Edu+ cells that costain with cone (Zpr1), MG (GS), amacrine cell (HuC/D in INL), and RGC (in RGC layer) antibodies; n = 3 different experiments. Error bars are SD. H, NMDA has no effect on Notch-responsive genes in the uninjured retina; n = 3 different experiments. Error bars are SD. I, DAPT-treatment inhibits MG proliferation in injured retinas expressing Ascl1 and Lin28a. Top illustration is timeline of experiment and graph quantifies the number of Sox9+ and BrdU+ double-labeled cells/retina; n = 5 different experiments. Error bars are SD. J, DAPT treatment has no effect on Notch-responsive genes in the uninjured AAV ShH10 Ascl1 and AAV ShH10 Lin28a transduced retina. n = 3 different experiments. Error bars are SD.