Figure 1.
Efficient loss of Otx2 gene products after self-knockout in adult retina.
A–C. Time course analysis of Otx2 gene products following tamoxifen injection at P30 in Otx2flox/CreERT2 mice. Relative levels of full-length (A) and total (normal + deleted) (C) Otx2 mRNA measured by RT-qPCR are shown. Corresponding levels of Otx2 protein (B) is shown in western blots. Position of size markers and Otx2 protein are indicated. Error bars in A and C are standard deviation. D. Staining of nuclei (DAPI) and Otx proteins on identical retina sections from control and mutant mice of the same genotype 4 days after tamoxifen injection. Shown are stitching of overlapping fields reconstituting a whole section. Right panel: magnification of boxed D′ and D″ areas. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer.
Figure 2.
Loss of Otx2 leads to photoreceptor cell degeneration.
A. Histology of control (non injected) and mutant (Tamoxifen administrated at P30) series of Otx2flox/CreERT2 retinas of the indicated ages. Sections are stained with Eosin and Haematoxylin. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B. Cell counts in the three layers retinal sections of control or tamoxifen treated (mutant) Otx2flox/CreERT2 mice at indicated ages. Normalized fields of the same eye area were used. Mean cell number and standard deviation are indicated for each condition (*P<0.001). C. Detection of apoptotic cells in control or tamoxifen treated Otx2flox/CreERT2 retinas 30 days after treatment. Left, middle and right panels show respectively DAPI staining, TUNEL labelled cells and superimposition of both images. D. Kinetics of apoptosis following Otx2 gene ablation. TUNEL labelled cells were counted on normalized sections of three independent mice for each stage, each corresponding to 0.063 mm2 of retinal area. Error bars are standard deviation.
Figure 3.
Consequences of Otx2 loss in photoreceptor cells.
A. Expression of cell type specific markers in P120 control (upper panels) and knockout (lower panels) retinas, following tamoxifen treatment at P30. B. Expression of cell type specific markers in P34 control (upper panels) and self-knockout (lower panels) retinas, following tamoxifen treatment at P30. C. Relative levels of Irbp mRNA in Otx2flox/CreERT2 retina following tamoxifen treatment. D. Relative levels of Crx mRNA in Otx2flox/CreERT2 retina following tamoxifen treatment. Error bars are standard deviation.
Figure 4.
Rapid induction of RPE disease by Otx2 self knockout.
A–I. Transmission electron microscopy images of control (A–C) and self-knockout Otx2flox/CreERT2 retina 10 days (D–F) and 20 days (G–I) after tamoxifen injection. Low (A,D,G), medium (B,E,H) and high (C,F,I) power views are presented, focusing on the area of interaction between RPE and photoreceptor disk-containing outer segments. Scale bars: 2 µm. J–K. Relative levels of Dct (J) and tyrosinase (K) mRNA in Otx2flox/CreERT2 retina following tamoxifen treatment. Error bars are standard deviation.