Fig. 3

9-HSA Affects RPC Proliferation by Activating Notch Pathway

(A–D) Expression analyses of the Notch target gene her4.2 at 2 and 3 days postfertilization (dpf) in DMSO-treated (respectively, (A) and (C)) and 9-HSA-treated retinae (respectively, (B) and (D)) reveal an increase in the expression of her4.2 in retinae of 9-HSA-injected embryos. At 2 dpf, her4.2 transcripts could still be found in the central part of the retinal neuroepithelium of 9-HSA-injected embryos while already restricted to the forming ciliary marginal zone (CMZ) in the control embryos (A) and (B). At 3 dpf, the expression of her4.2 was expanded in the CMZ in 9-HSA-treated retinae in comparison to the DMSO-injected embryos (C) and (D).

(E–H) Inhibition of the Notch pathway by DAPT treatment rescues 9-HSA’s differentiation phenotype on retinal ganglion cells (RGCs) at 2 dpf. Tg(atoh7:gap-RFP) DMSO-injected (E) and (G) and 9-HSA-injected (F) and (H) embryos were respectively treated with either a DMSO control solution or with a DAPT supplemented solution. DMSO-injected embryos treated with DAPT presented a thicker RGC layer. RGCs formed rosettes (white arrow) and a thicker optic nerve (F). DAPT treatment of 9-HSA-injected embryos resulted in the rescue of RGC differentiation (H) (n = 6 retinae for each condition). The average thickness of the RGC layer size was quantified for all conditions and we found that DAPT treatment increase significantly RGC layer thickness regardless of 9-HSA injection (in (G) versus (H): 10.31 μm ± 0.1643 in n = 3 and 30.22 μm ± 1.586 in n = 4, p value: 0.001, unpaired Student’s t test).

(I–K”) (I–J’) Intraocular injections of 9-HSA lead to an increase in proliferation of RPCs in the central part of the tissue compared to DMSO-injected retinae. 1 dpf embryos were injected intraocularly with 9-HSA in one eye and DMSO in the other eye (in n = 10 retinae). The injections were followed by an EdU incorporation assay at 2 dpf, and the effect of 9-HSA local retinal treatment on RPC proliferation was assessed at 3 dpf. Compared to the DMSO-injected eyes where almost all cells were EdU negative in the differentiated central part of the retina, retinae injected with 9-HSA still presented a high number of EdU-positive cells in this part of the tissue (K)–(K”). Intraocular injection of 9-HSA and DMSO in the right (R) and left (L) eye, respectively, of 2 dpf embryos followed by an in situ hybridization on her4.2 at 3 dpf. 9-HSA ocular injection leads to a striking increase of her4.2 expression in the central right retina and an expansion of its expression domain in the CMZ (K’) in comparison to DMSO-control-injected left retina (K”) (in n = 20 injected embryos).

Scale bars, 50 μm in (A)–(I), (J), and (K’)–(K”); 20 μm in (I’) and (J’); and 100 μm in (K).