Conditional RAX-Cre-mediated ablation of <i>Prss56</i> from fully differentiated Müller glia leads to ocular size reduction.

<p><i>Prss56</i> was conditionally ablated from Müller cells in a time-specific manner by crossing <i>Prss56</i>^<i>F/F</i> to the inducible RAX-Cre mouse strain (<i>Rax-Cre</i>^<i>ERT2</i>). CRE expression was induced by tamoxifen injection at P8, a time point preceding complete Müller glia differentiation. (<b>A</b>) Representative images of slit lamp examination by broad-beam illumination following tamoxifen injection at P8. <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes were indistinguishable from control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes at 2 months of age. (<b>B</b>, <b>C</b>) Following tamoxifen injection (TAM +; horizontal axis) at P8, <i>Prss56</i>^<i>F/F</i><i>Rax-Cre</i>^<i>ERT2</i> mice display a significant decrease in ocular axial length (<b>B</b>) and increase in retinal thickness (<b>C</b>) compared to control eyes (<i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> or <i>Prss56</i>^<i>F/F</i> mice without <i>Rax-Cre</i>^<i>ERT2</i>), N = 6 to 8 per group. (<b>D</b>) Following tamoxifen injection at P8, a significant reduction in VCD was detected in <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes compared to control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes, N = 6 per group. Ocular axial length, retinal thickness, and VCD in uninjected <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> and <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> mice were indistinguishable. (<b>E</b>) Representative OCT images showing reduced axial length and VCD in <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes compared to the control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> or <i>Prss56</i>^<i>F/F</i> mice. (<b>F</b>) qPCR analysis following tamoxifen injection at P8 revealed that <i>Prss56</i> mRNA was significantly upregulated in <i>Prss56</i>^<i>F/F</i> <i>; Rax-Cre</i>^<i>ERT2</i> retina compared to their <i>Prss56</i>^<i>F/+</i> <i>; Rax-Cre</i>^<i>ERT2</i> counterparts or uninjected controls, N≥ 6 per group. Values are presented as mean ±SD; * p<0.05, ** p<0.01, *** p<0.001, t-test. (<b>G-I</b>) Müller glia endfeet organization of <i>Prss56</i>^<i>Cre</i>; <i>R26</i>^{<i>tdTomato</i>} reporter mice during retinal development. Representative images of retinal section (<b>G, I</b>) or whole mount (<b>H</b>) showing Müller glia endfeet from control and <i>Prss56</i> mutant mice at P6. (<b>I</b>) Magnified images of the retinal endfeet are shown. The ILM of <i>Prss56</i> mutant mice (<i>Prss56</i>^{<i>Cre/gclr4</i>}; <i>R26</i>^{<i>tdTomato</i>}) at P6 is marked by regions of increased endfeet complexity (arrow head) compared to the ILM of control mice (<i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato</i>}). A substantial proportion of endfeet appear more spread out, occupying a larger area in the mutant compared to control retinal whole mounts (occupying smaller area). Red boxes highlight individual endfoot. N = 4 per genotype and scale bars = 17μm in <b>G</b> and <b>I</b>, and 50μm in <b>H</b>. ACD, anterior chamber depth; AL, axial length; VCD, vitreous chamber depth.