Phospho-Histone-3 and BrdU labeling show unexpected proliferation dynamics in the central retinas that have varying degrees of RGC loss.

<p>A–H. Representative confocal images at P0 and P4 show PH3-positive cells (green) in cryosections of wildtype and RGC-depleted retinas. I–L. Representative confocal images at P4 show PH3-positive cells in flat-mounted retinas. M–T. Representative confocal images at P0 and P4 show BrdU-positive cells (green) in cryosections. Red: PI. All scale bars  = 50 µm. U–W. Histograms comparing the numbers of proliferating cells estimated by PH3-positive cells in sections (U), PH3-positive cells in flat-mounts (V), and BrdU-positive cells in sections (W). No proliferating cells were found in the sampled region in all examined retinas at P7 and P10.</p

Assessment of apoptotic BPCs show a higher rate of apoptotic BPCs in the wildtype retina.

<p>A–L. Representative confocal images of the central retinas from wildtype, <i>Pou4f2^−/−, Atoh7^−/−</i>, and DKO mice at P5, P10 and P15. Retinas were labeled with antibodies to Chx10 (red), cleaved Caspase-3 (green) and TOPRO-3 (blue). Scale bar  = 50 µm. M. A histogram showing the percentage of Caspase-3 and Chx10-double positive cells over the Chx10-positive cells, depicting the ratio of apoptotic bipolar cells over all bipolar cells in four different retinas.</p

Birthdating experiments confirm fewer C-BPC and normal R-BPC production in the <i>Atoh7^−/−</i> retina.

<p>A, B. Representative confocal images of retinal cryosections from mice that received BrdU injection at P1 and harvested at P20. In the inner nuclear layer, Chx10 antibody (red) labels both R-BPCs and C-BPCs; PKCα antibody (green) labels only R-BPCs; and BrdU (blue) marks the cells born at P1. A′ and B′ show only the BrdU channel at a positions corresponding to A and B. Arrows indicate R-BPCs and arrowheads indicate C-BPCs. Scale bar  = 50 µm. Cells outside of the inner nuclear layer were not analyzed. Images of retinas with BrdU injected at other time points are not shown. C, D. Statistical analysis comparing C-BPC (C) and R-BPC (D) cell counts that were born on a specific day. Significantly fewer C-BPCs were born in the <i>Atoh7^−/−</i> retina on each examined day. In the sampling area, no C-BPCs were observed in either wildtype or <i>Atoh7^−/−</i> retinas born at P7.</p

Assessment of C-BPC and R-BPC numbers show reduced C-BPC numbers and normal R-BPC numbers in the RGC-depleted retinas.

<p>A–P. Representative confocal images from the central retinas from wildtype, <i>Pou4f2^−/−, Atoh7^−/−</i>, and DKO mice at P5, P10, P15, and P21. Samples were labeled with Chx10- (red) and PKCα- (green) to depict total BPCs and R-BPCs respectively. Nuclei were counterstained with TOPRO-3 (blue). Scale bar  = 50 µm. Q–S. Histograms comparing the mean numbers of total BPCs (Q), R-BPCs (R), and C-BPCs (S) in four different retinas with varying degrees of RGC depletion. Means of the C-BPC populations were calculated by taking the difference between Chx10- and PKCα- positive cell counts.</p

Phospho-Histone-3 and BrdU labeling of the peripheral retinas show the number of proliferating cells corresponds to the existing RGC number.

<p>A–H. Representative confocal images at P0 and P4 show PH3-positive cells (green) in cryosections of wildtype and RGC-depleted retinas. I–L. Representative confocal images at P4 show PH3-positive cells in flat-mounted retinas. Dash lines mark the edge of the retina. M–T. Representative confocal images at P4 and P7 show BrdU-positive cells (green) in cryosections. Red in all panels: propidium iodide. All scale bars  = 50 µm. U–W. Histograms comparing the numbers of proliferating cells estimated by PH3-positive cells in sections (U), PH3-positive cells in flat-mounts (V), and BrdU-positive cells in sections (W). Proliferating cell numbers found in all retinas at P10 are either zero or close to zero. There is no significant difference between any two genotypes at P10.</p

The number of <i>Vsx1</i>-expressing cells supports that fewer C-BPCs are found in the <i>Atoh7^−/−</i> retina.

<p>A–F. Confocal images of cryosections showing <i>Vsx1^LacZ/+</i>-expressing C-BPCs (green) in the wildtype (<i>Atoh7^+/−;Vsx1^LacZ/+</i>) and <i>Atoh7^−/−</i> (<i>Atoh7^−/−;Vsx1^LacZ/+</i>) retinas. Green: LacZ; red: propidium iodide. Scale bar  = 50 µm. G. A histogram comparing the mean number of <i>Vsx1^LacZ</i>-positive cells in wildtype and <i>Atoh7^−/−</i> retinas.</p

Experimental results with different algorithm.

<p>(a)original images. (b)ground truth. (c)the improved pre-extraction images. (d)previous percolation algorithm. (e) CrackForest. (f) CrackHHP.</p

Differentially expressed microRNAs based on clinico-pathological characteristics.

<p>^a The table only includes the miRNAs that were expressed differently between the corresponding two groups with significant p-values < 0.05.</p><p>^b miRNA levels were expressed as the mean±SD. <i>P</i> value was calculated by the Mann—Whitney U-test and was corrected by the Benjamini-Hochberg FDR test. The outcomes with FDR less than 0.05 were in bold.</p><p>Differentially expressed microRNAs based on clinico-pathological characteristics.</p

Divided image into several small grids with the overlapping regions.

<p>Divided image into several small grids with the overlapping regions.</p

Precision rate comparison.

<p>Precision rate comparison.</p