Figure 3
From: Zac1 functions through TGFβIIto negatively regulate cell number in the developing retina
Loss of Zac1 results in increased proliferation and reduced apoptosis at a late stage of retinogenesis. (a-d) BrdU labeling (red) of E18.5 wild-type and Zac1+m/- explants cultured 4DIV (a,b) or 6DIV (c,d). Arrowheads in (d) mark ectopic proliferating cells. (e) Percentage of BrdU+ nuclei in wild-type (black bar; E13.5: 2,824 BrdU+/8,235 DAPI+; E16.5: 2,234 BrdU+/10,663 DAPI+; E18.5: 2,859 BrdU+/27,380 DAPI+; E18.5→2DIV: 4,371 BrdU+/54,554 DAPI+; E18.5→4DIV: 988 BrdU+/55,300 DAPI+; E18.5→6DIV: 0 in 9 fields) and Zac1+m/- retinae (grey bars; E13.5: 3,555 BrdU+/10,413 DAPI+; E16.5: 3,369 BrdU+/15,707 DAPI+; E18.5: 2,212 BrdU+/17,642 DAPI+; E18.5→2DIV: 3,298 BrdU+/35,085 DAPI+; E18.5→4DIV: 3,474 BrdU+/97,499 DAPI+; E18.5→6DIV: 54 BrdU+/11,618 DAPI+). (f) BrdU-labeling indices of individual wild-type (squares) and Zac1+m/- (triangles) E18.5→4DIV retinal explants. (g,h) E18.5→4DIV wild-type (g) and Zac1+m/- (h) retinal explants labeled with CcnD1 (red). (i) Percentage of Ccdn1+ cells in wild-type (black bar; 2,480 CcnD1+/21,329 DAPI+) and Zac1+m/- without aberrant proliferation (grey bar; 3,156 CcnD1+/26,328 DAPI+) and with a proliferative phenotype (w/φ; white bar; 3,266 CcnD1+/18,709 DAPI+) at 4DIV. (j) Ccnd1-labeling indices of individual wild-type (squares) and Zac1+m/- (triangles) E18.5→4DIV retinal explants. (k,l) E18.5→4DIV wild-type (k) and Zac1+m/- (l) retinal explants labeled with pHH3 (red). (m) Apical (Ap) to basal (Ba) ratio of pHH3+ cells in wild-type (black bar; 808 ap:791 ba pHH3+) and Zac1+m/- without (grey bar; 971 ap:796 ba pHH3+) and with (w/φ; white bar; 1,012 ap:480 ba pHH3+) a proliferative phenotype at 4DIV. (n) Ap:Ba ratios of pHH3+ cells in individual wild-type (squares) and Zac1+m/- (triangles) E18.5→4DIV retinal explants. (o-p) Active caspase-3 (Ac-3) expression (red) in wild-type and Zac1+m/- E18.5→4DIV explants. Blue is DAPI counterstain. (q) Percentage of apoptotic cells in the total population of wild-type (black bars; E18.5: 71 ac-3+/18,341 DAPI+; E18.5→2DIV: 532 ac-3+/14,995 DAPI+; E18.5→4DIV: 1,266 ac-3+/27,321 DAPI+; E18.5→8DIV: 294 ac-3+/10,209 DAPI+) and Zac1+m/- (white bars; E18.5: 67 ac-3+/13,768 DAPI+; E18.5→2DIV: 457 ac-3+/13,195 DAPI+; E18.5→4DIV: 488 ac-3+/24,077 DAPI+; E18.5→8DIV: 212 ac-3+/14,377 DAPI+) retinae. (r) Distribution of individual wild-type (squares) and Zac1+m/- (triangles) ac-3-labeling indices at 4DIV.