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Cell-type specific roles for PTEN in establishing a functional retinal architecture.

Cantrup R, Dixit R, Palmesino E, Bonfield S, Shaker T, Tachibana N, Zinyk D, Dalesman S, Yamakawa K, Stell WK, Wong RO, Reese BE, Kania A, Sauvé Y, Schuurmans C - PLoS ONE (2012)

Bottom Line: Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period.Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.

Methodology/principal findings: In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.

Conclusions/significance: We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.

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Related in: MedlinePlus

Aberrant cellular mosaicism in Pten cKOs.(A–H) Immunolabeling of P21 wild-type (A) and Pten cKO (B) retinal flatmounts with TH. Voronoi diagrams depicting the distribution of TH+ amacrine cells in P21 wild-type (C) and Pten cKO (D) retinae. Calculation of TH+ Voronoi domain areas and their relative distributions in these two fields for P21 wild-type (C′) and Pten cKO (D′) retinae. Near neighbors of a TH+ reference cell in P21 wild-type (E) and Pten 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 Pten cKO (F′) retinae. Calculation of Voronoi domain (G) and Nearest Neighbor (H) regularity indices for TH+ amacrine cells in wild-type and Pten cKO retinae. (I–P) Immunolabeling of P21 wild-type (I) and Pten cKO (J) retinal flatmounts with calbindin. Voronoi diagrams depicting the distribution of calbindin+ horizontal cells in P21 wild-type (K) and Pten cKO (L) retinae. Calculation of TH+ Voronoi domain areas and their frequency distributions in P21 wild-type (K′) and Pten cKO (L′) retinae in these two fields. Near neighbors of a calbindin+ reference cell in P21 wild-type (M) and Pten cKO (N) retinae, with the nearest neighbour indicated in red. Frequency distribution of distances between TH+ amacrine cells in P21 wild-type (M′) and Pten 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 Pten cKO retinae. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 600 µm (A,B), 100 µm (C,D).
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pone-0032795-g003: Aberrant cellular mosaicism in Pten cKOs.(A–H) Immunolabeling of P21 wild-type (A) and Pten cKO (B) retinal flatmounts with TH. Voronoi diagrams depicting the distribution of TH+ amacrine cells in P21 wild-type (C) and Pten cKO (D) retinae. Calculation of TH+ Voronoi domain areas and their relative distributions in these two fields for P21 wild-type (C′) and Pten cKO (D′) retinae. Near neighbors of a TH+ reference cell in P21 wild-type (E) and Pten 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 Pten cKO (F′) retinae. Calculation of Voronoi domain (G) and Nearest Neighbor (H) regularity indices for TH+ amacrine cells in wild-type and Pten cKO retinae. (I–P) Immunolabeling of P21 wild-type (I) and Pten cKO (J) retinal flatmounts with calbindin. Voronoi diagrams depicting the distribution of calbindin+ horizontal cells in P21 wild-type (K) and Pten cKO (L) retinae. Calculation of TH+ Voronoi domain areas and their frequency distributions in P21 wild-type (K′) and Pten cKO (L′) retinae in these two fields. Near neighbors of a calbindin+ reference cell in P21 wild-type (M) and Pten cKO (N) retinae, with the nearest neighbour indicated in red. Frequency distribution of distances between TH+ amacrine cells in P21 wild-type (M′) and Pten 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 Pten cKO retinae. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 600 µm (A,B), 100 µm (C,D).

Mentions: To test whether Pten also regulates the tangential dispersion of retinal cells, we assessed the mosaic distribution of horizontal and amacrine cell subtypes in P21 retinal flatmounts. Anti-TH was used to label dopaminergic amacrine cells. In wild-type retinae, TH+ cell somata mosaics were dispersed in a patterned array (Figure 3A) [34]. In contrast, in Pten cKO retinas, TH+ amacrine cells appeared less regularly distributed (Figure 3B). To quantify the regularity of the cellular spacing in these mosaics, we examined their spatial properties using Voronoi domain and nearest neighbor analyses. Voronoi domain analysis computes the territory surrounding each cell that is closer to that cell than any of the neighboring cells [35]. Visual analysis of these Voronoi diagrams (Figure 3C,D) and of plots comparing the frequency of domain areas (Figure 3C′,D′) revealed a greater variability in domain sizes in Pten cKO retinae. To analyze this quantitatively, the Voronoi domain regularity index was calculated for each individual field (average domain area/standard deviation; Figure 3G), with higher values indicative of more regular spacing. As expected, TH+ regularity indices derived from this Voronoi tessellation were significantly lower in Pten cKO retinae (p<0.0001; Figure 3G, Table S2). Nearest neighbour analyses revealed a comparable increase in the variability of this measure (Figure 3E,F), as revealed by the skewed distribution of nearest neighbour distances for the TH+ amacrine cells in Pten cKO retina (Figure 3E′F′), resulting in a significant decrease in the nearest neighbor regularity index (p<0.0001; Figure 3H).


Cell-type specific roles for PTEN in establishing a functional retinal architecture.

Cantrup R, Dixit R, Palmesino E, Bonfield S, Shaker T, Tachibana N, Zinyk D, Dalesman S, Yamakawa K, Stell WK, Wong RO, Reese BE, Kania A, Sauvé Y, Schuurmans C - PLoS ONE (2012)

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

Show All Figures
getmorefigures.php?uid=PMC3293905&req=5

pone-0032795-g003: Aberrant cellular mosaicism in Pten cKOs.(A–H) Immunolabeling of P21 wild-type (A) and Pten cKO (B) retinal flatmounts with TH. Voronoi diagrams depicting the distribution of TH+ amacrine cells in P21 wild-type (C) and Pten cKO (D) retinae. Calculation of TH+ Voronoi domain areas and their relative distributions in these two fields for P21 wild-type (C′) and Pten cKO (D′) retinae. Near neighbors of a TH+ reference cell in P21 wild-type (E) and Pten 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 Pten cKO (F′) retinae. Calculation of Voronoi domain (G) and Nearest Neighbor (H) regularity indices for TH+ amacrine cells in wild-type and Pten cKO retinae. (I–P) Immunolabeling of P21 wild-type (I) and Pten cKO (J) retinal flatmounts with calbindin. Voronoi diagrams depicting the distribution of calbindin+ horizontal cells in P21 wild-type (K) and Pten cKO (L) retinae. Calculation of TH+ Voronoi domain areas and their frequency distributions in P21 wild-type (K′) and Pten cKO (L′) retinae in these two fields. Near neighbors of a calbindin+ reference cell in P21 wild-type (M) and Pten cKO (N) retinae, with the nearest neighbour indicated in red. Frequency distribution of distances between TH+ amacrine cells in P21 wild-type (M′) and Pten 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 Pten cKO retinae. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. Scale bars = 600 µm (A,B), 100 µm (C,D).
Mentions: To test whether Pten also regulates the tangential dispersion of retinal cells, we assessed the mosaic distribution of horizontal and amacrine cell subtypes in P21 retinal flatmounts. Anti-TH was used to label dopaminergic amacrine cells. In wild-type retinae, TH+ cell somata mosaics were dispersed in a patterned array (Figure 3A) [34]. In contrast, in Pten cKO retinas, TH+ amacrine cells appeared less regularly distributed (Figure 3B). To quantify the regularity of the cellular spacing in these mosaics, we examined their spatial properties using Voronoi domain and nearest neighbor analyses. Voronoi domain analysis computes the territory surrounding each cell that is closer to that cell than any of the neighboring cells [35]. Visual analysis of these Voronoi diagrams (Figure 3C,D) and of plots comparing the frequency of domain areas (Figure 3C′,D′) revealed a greater variability in domain sizes in Pten cKO retinae. To analyze this quantitatively, the Voronoi domain regularity index was calculated for each individual field (average domain area/standard deviation; Figure 3G), with higher values indicative of more regular spacing. As expected, TH+ regularity indices derived from this Voronoi tessellation were significantly lower in Pten cKO retinae (p<0.0001; Figure 3G, Table S2). Nearest neighbour analyses revealed a comparable increase in the variability of this measure (Figure 3E,F), as revealed by the skewed distribution of nearest neighbour distances for the TH+ amacrine cells in Pten cKO retina (Figure 3E′F′), resulting in a significant decrease in the nearest neighbor regularity index (p<0.0001; Figure 3H).

Bottom Line: Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period.Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.

Methodology/principal findings: In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.

Conclusions/significance: We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.

Show MeSH
Related in: MedlinePlus