Abnormal patterning of the hypertrophic IPL in <i>Pten</i> cKOs.

<p>(A–D) P21 wild-type and <i>Pten</i> cKO retinae labelled in wholemount for TH (A,B) and melanopsin (C,D). Arrowheads in B,D mark increased fasciculation of TH⁺ amacrine cell processes and melanopsin⁺ RGC dendrites in <i>Pten</i> cKO retinae, respectively. (E–H) P7 wild-type and <i>Pten</i> cKO retinae labelled with GFP (green; from <i>Pax6::cre</i> transgene) and Pax6 (red; E,F) or syntaxin (G,H). Bracket in F marks area where <i>Pax6::cre</i> transgene is not expressed. (I–T) P21 wild-type and <i>Pten</i> cKO retinae labelled with syntaxin (I,J), melanopsin (K,L), TH (M,N), calretinin (O,P), ChAT (Q,R) and PKCα (S,T). gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; onl, outer nuclear layer; opl, outer plexiform layer; re, retina. Scale bars = 50 µm (A,B,G,H,K–T), 100 µm (C,D,I,J), 600 µm (E,F).</p

PTEN retinal expression and generation of retinal-specific <i>Pten</i> cKO.

<p>(A–E) Co-labeling of P7 retina with PTEN (red) and Brn3b (green, A), calbindin (green, B), Pax6 (green, C), Chx10 (green, D) and rhodopsin (green, E). Blue is DAPI counterstain. Insets to the right of each panel are high magnification images of PTEN⁺ cells, showing co-expression in Brn3b⁺ RGCs (A), calbindin⁺ horizontal cells (B) and Pax6⁺ amacrine cells (C). Insets in D show low levels of PTEN co-expression in Chx10⁺ bipolar cells, while PTEN protein was not detected in rhodopsin⁺ rod photoreceptors (E). (F–I) Schematic illustration of crosses between transgenic animals carrying a Z/AP dual reporter and <i>Pax6</i> α-cre/P0 promoter::<i>Cre-IRES-GFP</i> transgene (hereafter designated <i>Pax6::Cre</i>) (F). Analysis of AP histochemical stain (i.e., recombined cells) in <i>Pax6::Cre</i>;Z/AP double transgenics at E12.5 (G) and in a P7 retinal flatmount (H). β-galactosidase histochemical stain (i.e., non-recombined cells) in a P7 retinal flatmount (I). Inset in G is a high magnification image of the eye, with the asterisk designating a lack of recombination in the central retina. (J) Schematic illustration of crosses between mice carrying a floxed <i>PTEN</i> allele (<i>PTEN^fl</i>) and <i>Pax6::Cre</i> transgene. (K–M) Expression of PTEN in P7 <i>Pten</i>^+/+ (K) and <i>Pten</i> cKO (L) retinal sections. Bracket in L shows central retina where <i>Pten</i> is not deleted and expression is maintained. Insets in K and L show PTEN immunolabeling of retinal flatmounts, confirming that PTEN expression is retained in the central retina in <i>Pten</i> CKOs. PTEN Western blot analysis and densitometry on P21 wild-type and <i>Pten</i> heterozygous and homozygous cKO retinae (M). (N,O) Expression of pAkt^Ser473 in P7 wild-type (N) and <i>Pten</i> cKO (O) retinae. Asterisks in O mark aberrant aggregations of amacrine dendrites in the INL. (P,Q) Western blot analysis and densitometry of pAkt^Ser473/Akt (P) and pS6^Ser235/236/S6 (Q) in P21 wild-type and <i>Pten</i> cKO retinal lysates. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; on, optic nerve; onbl, outer neuroblast layer; onl, outer nuclear layer; opl, outer plexiform layer; re, retina. Scale bars = 50 µm (A–E,N,O), 2 mm (G), 1 mm (H,I), 600 µm (K,L).</p

Abnormal retinal architecture and increased retinal cell sizes in <i>Pten</i> cKOs.

<p>(A–D) Low (A,B) and high (C,D) magnification images of hematoxylin-eosin (H&E) stained sections of adult wild-type (A,C) and <i>Pten</i> cKO (B,D) retinae. (E–G) Calbindin labelling of P7 wild-type and <i>Pten</i> cKO retinal sections (E,F) and area measurements of calbindin⁺ horizontal cells (G). (H–S) Labeling of retinal flatmounts from P21 wild-type and <i>Pten</i> cKOs with calbindin (H,I), ChAT (K,L), TH (N,O), and SMI32 (Q,R). Calculation of cell areas for P21 calbindin⁺ horizontal cells (J), ChAT⁺ (M) and calbindin⁺ (P) amacrine cells and SMI32⁺ RGCs (S). p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 100 µm (A,B,N,O), 50 µm (C–L,Q,R).</p

Interactions between <i>Pten</i> and <i>Dscam</i>.

<p>(A,B) Distribution of <i>Dscam</i> transcripts in P7 wild-type (A) and <i>Pten</i> cKO (B) retinae. (C–F) Labeling of E18.5 wild-type and <i>Dscam</i> KO retinae with Pax6 (red)/syntaxin (green; C,D) and calretinin (red; E,F). Blue is DAPI counterstain. (G–I) Western blotting and densitometry for PTEN and pPTEN^Ser380 (G), total Akt and pAkt^Ser473 (H), and total S6 and pS6^Ser235/236 (I) in E18.5 wild-type and <i>Dscam</i> mutants. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; opl, outer plexiform layer. Scale bars = 50 µm.</p

Aberrant cellular mosaicism in <i>Pten</i> cKOs.

<p>(A–H) Immunolabeling of P21 wild-type (A) and <i>Pten</i> cKO (B) retinal flatmounts with TH. Voronoi diagrams depicting the distribution of TH⁺ amacrine cells in P21 wild-type (C) and <i>Pten</i> cKO (D) retinae. Calculation of TH⁺ Voronoi domain areas and their relative distributions in these two fields for P21 wild-type (C′) and <i>Pten</i> cKO (D′) retinae. Near neighbors of a TH⁺ reference cell in P21 wild-type (E) and <i>Pten</i> cKO (F) retinae, with the nearest neighbour indicated in red. Frequency distribution of nearest neighbor distances between TH⁺ amacrine cells in these two fields for P21 wild-type (E′) and <i>Pten</i> cKO (F′) retinae. Calculation of Voronoi domain (G) and Nearest Neighbor (H) regularity indices for TH⁺ amacrine cells in wild-type and <i>Pten</i> cKO retinae. (I–P) Immunolabeling of P21 wild-type (I) and <i>Pten</i> cKO (J) retinal flatmounts with calbindin. Voronoi diagrams depicting the distribution of calbindin⁺ horizontal cells in P21 wild-type (K) and <i>Pten</i> cKO (L) retinae. Calculation of TH⁺ Voronoi domain areas and their frequency distributions in P21 wild-type (K′) and <i>Pten</i> cKO (L′) retinae in these two fields. Near neighbors of a calbindin⁺ reference cell in P21 wild-type (M) and <i>Pten</i> cKO (N) retinae, with the nearest neighbour indicated in red. Frequency distribution of distances between TH⁺ amacrine cells in P21 wild-type (M′) and <i>Pten</i> cKO (N′) retinae in these two fields. Calculation of Voronoi domain (O) and Nearest Neighbor (P) regularity indices for calbindin⁺ horizontal cells in wild-type and <i>Pten</i> cKO retinae. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 600 µm (A,B), 100 µm (C,D).</p

Altered ERG oscillatory potential responses in <i>Pten</i> cKO animals.

<p>(A–F) Scotopic ERG with representative trace (A; wild-type is black; <i>Pten</i> cKO is red) and OP scalogram (B,C) at flash intensity of 0.38 cd*s/m². (D–F) Graphical representation of OP amplitude (D), frequency (E) and latency (F) across 19 steps (−5.22 to 2.86 cd*s/m²). (G–L) Double flash ERG with representative trace (G; wild-type is black; <i>Pten</i> cKO is red) and OP scalogram (H,I) at flash intensity of 0.38 cd*s/m². (J–L) Graphical representation of OP amplitude (J), frequency (K) and latency (L) across 10 steps (−5.22 to 2.86 cd*s/m²).</p

Synaptic contacts in the <i>Pten</i> cKO retinal IPL.

<p>(A–F) Electron microscopy (EM) of adult wild-type and <i>Pten</i> cKO retinae. Schematic illustration of retinal architecture (A′). Low magnification EM images of wild-type (A) and <i>Pten</i> cKO (B) retinae, shown to scale, illustrating expansion of mutant retinae. Higher magnification images of <i>Pten</i> cKO IPL (C–F), with boxed areas in C shown in higher magnification in D,E. Asterisks in C mark ectopic cells in the IPL. Color scheme in D–F′ is as follows: Blue denotes rod bipolar cell terminal with ribbons (labeled R) in the <i>Pten</i> cKO IPL (D). Pink denotes amacrine cell synapses on ectopic somata within the IPL (E,F). GCL, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; opl, outer plexiform layer. Scale bars = 10 µm (A,B,C), 1 µm (D,E), 2 µm (F), 100 µm (G–J), 50 µm (G′–J′).</p

Aberrant RGC fasciculation and subcortical visual responses in <i>Pten</i> cKOs.

<p>(A–B) Low (A,B) and high (A′,B′) power photomicrographs of SMI-32 labeling of P21 wild-type and <i>Pten</i> cKO retinal wholemounts. (C–E) Photomicrographs of wild-type and <i>Pten</i> cKO P21 optic nerves (C) and corresponding cross sections stained with hematoxylin-eosin (E). Optic nerve diameters are shown in D. (F,G) AP staining of P21 <i>Pax6::Cre</i>⁺;Z/AP⁺ (“wild-type”; F) and <i>Pten^f</i>^l/fl;<i>Pax6::Cre</i>⁺;Z/AP⁺ (<i>Pten</i> cKO, G) whole brains with the overlying cortex removed to reveal the visual pathway. The center of the superior colliculus (SC) is unstained as it is innervated by RGCs in the central retina, where cre activity is low. (H,I) Behavioural measures of the optokinetic reflex in adult wild-type and <i>Pten</i> cKOs that are either pooled (H) or separated into affected and unaffected groups (I). Scale bars = 300 µm (A,B), 100 µm (A′,B′), 750 µm (C), 200 µm (E), 2.5 mm (F,G).</p