Fig. 2

TPPP3 is expressed in mouse RGCs and promotes RGC neurite outgrowth ex vivo. A Western blots show that RGC markers BRN3A, RBPMS, and THY1 are expressed selectively in the immunopurified RGC cell population, as is Tppp3. B Immunostaining of RGCs for BRN3A and TPPP3 in adult mouse retinal sections reveals that Tppp3 is expressed within the RGC layer. ~ 75% of BRN3A+ cells co-express Tppp3 (white arrows). Scale bar = 100 µm. C Immunostaining of P2 primary RGCs shows expression of anti-β-III-tubulin antibody E7. Co-labeling with anti-β-III-tubulin antibody E7 and Tppp3 confirmed that Tppp3 is expressed primarily within the soma of RGCs. Scale bar = 50 µm. D RNAscope analysis of Tppp3 in the developing mouse eye. Tppp3 expression reached its peak at E14.5 and subsequently decreased. E Representative images of primary RGCs transduced with AAV2-control or AAV2-Tppp3-OE vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 overexpression increases RGC neurite outgrowth by ~ 20% (n = 5 independent cultures). F Representative images of primary RGCs transduced with AAV2-shCtrl or AAV2-shTppp3 vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 knockdown decreases neurite outgrowth by ~ 20%. Scale bar = 100 µm. Each data point reflects an independent cell culture. Statistical significance was determined using one sample t-test (****p < 0.0001, ***p < 0.001). Mean ± SEM is shown