Scleral modifications in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant eyes.

<p>Nissl staining of retinal sections in control (<b>A</b>) and mutant (<b>B</b>) eyes shows reduced scleral thickness at P90. The choroid and occasionally the RPE appear thicker at the posterior pole of the mutant eyes. Transmission electron micrographs of the posterior sclera wall (<b>C, D</b>) shows that the collagen fibrils form well-organized lamellae in the control sclera (<b>C</b>); in the mutant fibril-poor areas and impaired packing are evident (<b>D</b>). Contrary to the control sclera the collagen fibril density is lower in all layers of the mutant sclera (<b>E</b>). Transmission electron micrographs showed fibril collagen organization within a lamella of the posterior sclera (<b>F, H</b>) and in localized areas (<b>G, I</b>) of control (<b>F, G</b>) and mutant (<b>H, I</b>). Fibrils were morphologically abnormal with irregular contours and heterogeneous diameters in the mutants. Measurements of cross-sectional diameters of fibrils from the inner, middle and outer posterior sclera in control (<b>J</b>) and mutant eyes (<b>K</b>). The mean fibril diameter distribution is modified in the mutants in all three layers; the frequency of very small as well large diameter fibrils is increased. (ch) choroid, (fb) fibroblast, (Lg) longitudinal and (Tr) transversal orientation of the cross-sectioned fibrils. Two-tailed unpaired <i>t</i> test was used. ***<i>P</i><0.001, Values are mean ± SEM of 3 animals per genotype; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129518#sec009" target="_blank">methods</a>. Scale bars: 50 μm in A, B; 3.5 μm in C, D; 1.2 μm in F, H; 300 nm in G, I.</p

Lrp2-deficient eyes are abnormally enlarged.

<p>Sagittal cryosections through the developing eye (<b>A-D</b>). Lrp2 is expressed in the developing neuroretina (nr) at E12.5 (<b>A</b>). From E15.5 onward the signal is restricted in the lens (L) facing inner layer of the ciliary body (cb) epithelium, a low expression is also seen in the outer layer of the CB and in the retinal pigmented epithelium (rpe) (<b>B</b>). Loss of Lrp2 signal in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants at E12.5 and E15.5 (<b>C, D</b>). Sagittal cryosections of control and mutant retinas at P60 showing retinal thinning and the presence of a posterior staphyloma in the mutant (<b>E</b>, <b>F</b>). Reconstruction of the mouse face using MRI at P60 (<b>G</b>, <b>H</b>). The <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants display bilateral eye enlargement, through the anterior-posterior axis and the equatorial diameter (double headed arrows in <b>G, H</b>). Horizontal MRI images showed that the retrobulbar space, between the orbit and the eyeball, (double-headed arrows in <b>I, J</b>) is decreased in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants. The corpus callosum (arrow in <b>I</b>) is not formed in the mutants (asterisk in J). (vz) ventricular zone. Scale bars: 25 μm in A-D; 600 μm in E, F.</p

Opthalmological evaluation of control and mutant mice.

<p>Similar gross morphology of the anterior segment in control (<b>A, A’</b>) and <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant littermates at P90 (<b>B, B’</b>). In some cases pupillary ectopia was observed in the mutants (<b>C, C’</b>). Fundus photographs of control (<b>D, F</b>) and mutant eyes (<b>E, G</b>) at P60 (<b>D, E</b>) and P180 (<b>F, G</b>) respectively show chorioretinal atrophy and peripapillary staphyloma. (<b>H</b>) Comparisons of intraocular pressure between control and mutant littermates at the indicated postnatal ages. Two-way ANOVA post hoc Tukey test was used, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 10 animals per age and genotype.</p

MRI analysis of post-natal eye growth in control and <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants over the first year of life.

<p>High resolution sagittal slices through the optical axis of the right eye of each mouse were acquired (<b>A-H</b>). A variety of optical parameters were extracted from the MRI images collected from groups of control and mutant mice at the ages indicated (<b>I-N</b>). Growth rate of axial length and vitreous chamber depth (<b>O</b>, <b>P</b>). A two-way ANOVA post hoc Tukey test was used, <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 4 animals per age and genotype. Scale bars: 1500 μm in A-H.</p

Decreased retinal cell density is associated with increased cell death in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant eyes.

<p>Reduced retinal cell density in the ONL, INL and GCL between P5 and P90. Values are expressed in % of normal thickness of the corresponding layers. Comparisons were calculated between P3 as 100% cell density and the other time points in each layer. Two-way ANOVA post hoc Tukey was used, ***<i>P</i><0.001, ns: non statistically significant, n = 5 animals per age. (<b>A</b>). The PH3 + cells are similarly distributed in control (<b>B</b>) and mutant retinal layers at P3 (<b>C</b>). Similar proliferation indexes in control and mutant retinas between E13.5 and P1, a significant reduction is seen in the mutants at P3 and P5 (<b>D</b>). In normal retinas TUNEL + cells are essentially seen in the INL and to a lesser extent in the ONL and GCL layers (<b>E</b>, upper panel). In the mutants the TUNEL signal appears stronger in the INL, ONL and IPL (<b>E</b>, lower panel). Cell death is significantly increased in the mutants between P5 and P21 (<b>F</b>). Distribution of caspase 3+ apoptotic cells in control and mutant retinas at P7 and P21 (<b>G</b>). Apoptosis is significantly increased in the mutants (<b>H</b>). The expression of the marker of autophagy Hsp70 is particularly strong in the mutant INL (<b>I</b>). Western-blot analysis of the indicated autophagic markers; GAPDH is used as an internal loading control (<b>J</b>). Two-way ANOVA post hoc Tukey test was used, **<i>P</i><0.01, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 5 animals per age and genotype; ***p<0.01. Scale bars: 50 μm in B, C, I; 30 μm in E, G.</p