Fig. 2

9-HSA Exogenous Administration Leads to Defects in Retinal Progenitor Cell Differentiation and to an Increase in Proliferation in the Retina

(A–D’) 9-HSA exogenous administration leads to defects in differentiation during retinal development for all cell types analyzed. (A)–(A’) Immunohistochemistry of anti-Zn5 (green) labeling retinal ganglion cells (RGCs) on 2 days post-fertilization (dpf) frontal retinal cryosections from DMSO-treated (A) and 9-HSA-treated (A’) embryos. No RGC labeling was observed in the central part of the retina at 2 dpf in 9-HSA treated in comparison to DMSO-treated control retinae. (B)–(B’) Immunohistochemistry of anti-Parvalbumin (green) labeling amacrine cells (AC) and displaced AC on 3 dpf frontal retinal cryosections from DMSO-treated (B) and 9-HSA-treated (B’) embryos. Similar to RGCs, no ACs or displaced ACs were observed in 9-HSA-treated in comparison to DMSO-treated control retinae. (C)–(C’) Immunohistochemistry of anti-GS (green) labeling Müller glia cells (MGs) on 3 dpf frontal retinal cryosections from DMSO-treated (C) and 9-HSA-treated (C’) embryos revealed no MG differentiation in 9-HSA-treated in comparison to the DMSO control retinae. (D)–(D’) Immunohistochemistry of anti-Zpr1 (green) labeling photoreceptor cells (Ph) on 3 dpf frontal retinal cryosections from DMSO-treated (D) and 9-HSA-treated (D’) embryos. No Zpr1 labeling was observed in the central part of 9-HSA-treated retinae compared to DMSO-treated control retinae, where Zpr1-positive Ph could be detected within the outer nuclear layer of the 3 dpf retinal tissue. Asterisk in (D’) indicates unspecific antibody trapping in the lens.

(E–I) 9-HSA exogenous administration leads to an increase in proliferation in the retina. (E)–(E’) EdU incorporation assay at 2.5 days postfertilization (dpf) in DMSO-treated control and 9-HSA-treated embryos detected by immunohistochemistry after 24 h shows an increase in the number of cells that actively replicated their DNA (S phase, red) during these 24 h at 3.5 dpf in the ciliary marginal zone, which was expanded in the 9-HSA-treated retinae. Nuclear counterstaining was also performed on these retinal sections by DAPI labeling (blue). (F)–(F’) Immunohistochemistry of anti-PCNA (red) labeling cells in S phase of the cell cycle on 3 dpf frontal retinal cryosections. 9-HSA-treated retinae showed an expanded PCNA labeling in the ciliary marginal zone and in the central retina (white arrow) in comparison to DMSO-treated control retinae at 3 dpf. (G) Quantification of the cell-cycle duration between two S phases of PCNA-GFP-positive RPCs from 9-HSA- and DMSO-injected embryos from 32 to 48 hpf. Time was measured in min. A significant increase in the cell-cycle length of RPCs from 9-HSA-injected embryos was observed (345.5 ± 23.24 min for DMSO in n = 13 cells compared to 691.3 ± 68.06 min for 9-HSA in n = 8 cells, p value: 0.0003 depicted as ^∗∗∗). Statistical significance was determined using a Mann-Whitney U test. (H–H’) Immunohistochemistry of anti-pH3 (green) labeling of cells in M phase of the cell cycle on 3 dpf frontal retinal cryosections counterstained with the nuclear marker DAPI (blue). 9-HSA-treated retinae showed a higher number of mitotic cells in comparison to DMSO-treated control retinae at 3 dpf. (I) Quantification of the mitotic index at 2 and 3 dpf shows a significant increase in the percentage of pH3-positive cells in 9-HSA-treated retinae with respect to DMSO-treated control retinae at 2 and 3 dpf (n = 5, p value: 0.0176 at 2 dpf and n = 4, p value: 0.0060 at 3 dpf). Statistical significance for this quantification was determined using Student’s t test and depicted as: ^∗p value < 0.05; ^∗∗ p value < 0.01. Scale bars, 50 μm.