Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
Figure 3

Figure 3. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Decreased Müller glia numbers in Pten-cko retinas. The distribution of neuronal subtypes in P8 Pax6-aCre and Pten-cko mouse retinas was examined using the cell type-specific markers, Brn3b (A, B), for RGCs; Pax6 (C, D), for ACs; Prox1 (E, F), for HZs (arrowheads) and ACs; Chx10 (G, H), for BPs; Crx (I, J), for PRs; and Sox9 (K, L), for MGs. (M) Cell composition of Pax6-aCre (open bars) or Pten-cko (filled bars) littermate mouse retinas is shown. The numbers represent mean values obtained from four different retinal samples isolated from two independent litters. Error bars denote s.d. *P-value <0.01; **P-value <0.005. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 μm.

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
2.
Figure 7

Figure 7. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Pten plays a pivotal role in retinal neurogenesis by supporting temporal activation of Notch signalling in RPC. Pten supports Notch signalling by negatively regulating Akt that inactivates NICD transcription complex formation. The accumulated Notch transcription activator complex composed of CBF1, NICD, MAM, and CBP/p300 then induces the expression of downstream target genes including transcription repressor Hes1, one of which downstream target is Pten, to prevent RPCs from prematurely differentiating. As a result of Hes1-induced repression of Pten, Akt is reactivated to turn the Notch-induced transcriptions off, and consequently RPCs start to differentiate. Upon the disassembly of Notch transcription activator complex, Hes1 cannot be accumulated to repress Pten expression, and re-expressed Pten start to inhibit Akt. This feedback regulation loop coordinates retinal neurogenesis and RPC maintenance (A). In Pten-deficient RPCs, this feedback loop is broken. Consequently, the NICD fails to form transcription activator complex that is necessary for RPC maintenance (B).

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
3.
Figure 2

Figure 2. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Premature depletion of hyperproliferating Pten-deficient RPCs. (A) The proliferating cells in P0 and P4 retinas were detected by immunostaining with an anti-BrdU antibody (see details in Materials and methods). (B) The BrdU-positive cells in the proximal and distal retina at distinct developmental stages were counted. The open squares and open triangles indicate percentage of BrdU-positive cells in distal and proximal areas of the Pax6-aCre retina, respectively, whereas the closed squares and solid triangles indicate percentage of BrdU-positive cells in the distal and proximal areas of the Pten-cko retina. Y axis values in the graph are the mean value obtained from six (E16.5 and P0 samples) or eight (P4 and P8 samples) different samples taken of retinal sections isolated from three (E16.5 and P0 samples) or four (P4 and P8 samples) independent litters. Error bars denote standard deviation (s.d.). *P-value <0.01; **P-value <0.005. (C) The distribution of Sox2-positive RPCs in P4 mouse retinas was examined by immunostaining with an anti-Sox2 antibody. (D) The mean values of Sox2-positive RPCs, obtained from data of six different images of eye sections from three independent litters, are displayed. Error bars denote s.d. **P-value <0.005. Scale bars in (A) and (C) indicate 50 μm. Dashed lines in the pictures represent retina-RPE borders.

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
4.
Figure 6

Figure 6. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Ectopic NICD rescues developmental defects in Pten-cko retina. (A) The NICD was ectopically expressed in Pax6-aCre and Pten-cko mouse retinas upon Pax6-aCre-mediated excision of loxP-STOP-loxP gene cassette (Murtaugh et al, 2003). The decreased mitotic index, which was measured by counting the number of pH3-positive cells (top row), in P4 Pten-cko retina was recovered to a greater extent by ectopic expression of NICD than that of littermate Pax6-aCre retina. Sox2-positive RPCs (middle row), which were depleted in P4 Pten-cko retinas, also increased upon expression of NICD, whereas the number of prematurely differentiated Rhodopsin-expressing rPRs (bottom row, arrowheads) in Pten-cko retinas was decreased to normal levels. The dashed lines in the pictures denote the borders between retina and RPE. Scale bars, 50 μm. (B) Graph presents cells, which are positive to pH3, Sox2, or Rhodopsin in retinal sections of P4 Pax6-aCre, Pax6-aCre;R26-NICD, Pten-cko, or Pten-cko;R26-NICD littermate mice, including the images shown in (A). The values are averages in five different images obtained from three independent litters. Error bars are s.d. *P-value <0.005; **P-value <0.001 (Student's t-test). P-values for each marker obtained by one-way ANOVA test were <0.005 (results not shown).

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
5.
Figure 4

Figure 4. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Accelerated development of Pten-cko retinas. (A) The distribution of mature forms of retinal neurons in developing Pax6-aCre and Pten-cko littermate mouse retinas was examined by immunostaining with antibodies against Brn3b, Calbindin, Recoverin, Glutamine synthase (GS), and Protein kinase C-α (PKCα). The time point examined for each cell type corresponds to the time at which the first signals are detectable. Asterisks (*) in GS staining images represent non-specifically stained cells. Scale bars, 50 μm. (B) Retinal cells expressing each marker were counted and mean numbers of positive cells per 1000 total cell counts are shown in Y axis. The values were obtained from six different immunostaining images of retinal sections, which were obtained from two independent litters. Error bars are s.d. *P-value <0.01; **P-value <0.005. (C) The distribution of post-mitotic neurons in E12.5 retinas was assessed by immunostaining with a neuron-specific class III β-tubulin antibody (Tuj1; red) and counterstaining with the RPC-specific markers Pax6-aEGFP (green). More Tuj1-positive post-mitotic neurons were EGFP positive in Pten-cko retinas (arrows) than in Pax6-aCre retinas, in which Tuj1 and EGFP positivity were mutually exclusive. Dashed boxes indicate areas where EGFP-positive RPCs and Tuj1-positive post-mitotic neurons intermingled. (D) The cells positive to Tuj1 or double positive to Tuj1 and Pax6-aEGFP were counted. The Y axis values are mean values obtained from five independent samples. Scale bars, 50 μm. Error bars are s.d. **P-value <0.005.

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
6.
Figure 1

Figure 1. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Specific enrichment of Akt activity in post-mitotic retinal neurons. (A) The activity of PI3K-Akt signalling in the developing mouse retina was examined by co-immunostaining using a rabbit anti-phospho-Akt[S473] antibody (pAkt; green) that recognizes activated Akt, and guinea pig antibodies that detect Chx10 expression in RPCs (red in top row) or Islet1 in post-mitotic neurons (red in bottom row). Arrowheads indicate Chx10-positive RPCs that lack pAkt (top row) and Islet1-positive post-mitotic neurons that co-express pAkt (bottom row), respectively. (B) Schematic diagram for spatial-specific elimination of Pten gene (red) in mouse retina using Pten-flox and Pax6-aCre (green) mice (Marquardt et al, 2001; Suzuki et al, 2001). (C) Corresponding to the Pten elimination, pAkt level was significantly increased in Pten-cko mouse retina. Arrows and arrowheads at top row indicate Pten(+)/EGFP(−) and Pten(+)/EGFP(+) cells, respectively. Arrowheads at bottom row indicate pAkt-positive cells. *Pten expressed in extra-retinal cells. (D) Distribution of Pten-deficient cells in E14.5 Pten-cko mouse retina was indirectly examined by X-gal staining (blue) that detects β-galactosidase expressed by R26-lacZ reporter (R26-lacZ) gene cassette upon the Pax6-aCre-dependent recombination. The area that was unaffected by the Pax6-aCre-dependent recombination (X-gal-negative inside area of the cone) was reduced to 46° angle in E14.5 Pten-cko;R26R retina from 83° angle at the equivalent position of littermate Pax6-aCre;R26R retina. RBL, retinoblast layer; GCL, ganglion cell layer. Scale bars in the pictures represent 50 μm. Dashed lines in the pictures represent retina-RPE borders.

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.
7.
Figure 5

Figure 5. From: Pten coordinates retinal neurogenesis by regulating Notch signalling.

Reduced Notch signalling in Pten-cko mouse retina. (A) The expression of the Notch target Hes1 in E14.5 Pax6-aCre and Pten-cko mouse retina was examined by immunostaining with an anti-Hes1 antibody (red). Co-immunostaining with an anti-pAkt antibody (blue) revealed an inverse correlation between Hes1 expression and Akt activity. (B) Images are magnified versions of the boxed areas in (A). Arrows and arrowheads indicate pAkt- and Hes1-positive cells, respectively. (C) Neonatal (P0) retinal cell lysates (including 50 μg proteins/well) were analysed by western blotting using an anti-Notch1 antibody that recognizes the C-terminal region of mouse Notch1 (top panel of left column). The arrowheads in the top panel indicate full-length Notch1 (N1-FL); extracellular domain truncated Notch1 fragments with transmembrane domain and cytoplasmic domain (N1-TM). Asterisk (*) indicates non-specific proteins recognized by anti-Notch1 antibody. Notch1 intracellular domain (NICD) was detected by anti-NICD antibody to analyse γ-secretase-mediated cleavage of Notch1 (bottom panel of left column). The successful elimination of Pten and consequent hyperactivation of Akt in the retinal cells were confirmed by western blotting with anti-Pten, pAkt, and anti-Akt antibodies, respectively (right column). The amount of proteins in the lysates was normalized by the amount of β-actin. (D) The P0 Pax6-aCre and Pten-cko mouse retinal lysates (including 2 mg proteins) were subjected to immunoprecipitation with anti-NICD antibody (2 μg), and co-immunoprecipitated CBF1 and MAML1 proteins were detected by western blotting with anti-CBF1 or anti-MAML1 antibodies (see details in Materials and methods). rbIgGH, rabbit immunoglobulin G heavy chain. The relative amounts of CBF1 and MAML1 in the cell lysates (including 50 μg proteins/well) were also examined by western blotting. Relative band intensity values in (C) and (D) were obtained by Image-J densitometry analysis program. Information for the antibodies is provided in Supplementary Table S2. Figure source data can be found in Supplementary data.

Hong Seok Jo, et al. EMBO J. 2012 February 15;31(4):817-828.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk