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Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells.

Wong KA, Trembley M, Abd Wahab S, Viczian AS - Biol Open (2015)

Bottom Line: We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos.We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone.In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA The Center for Vision Research, SUNY Eye Institute, Upstate Medical University, Syracuse, NY 13210, USA.

No MeSH data available.


Related in: MedlinePlus

Dual inhibition of Smad1/5/8 and Smad2 activity with chemical inhibitors DM and SB43 is sufficient to drive retinal formation.Embryos were injected with Smad2 (50 pg), Noggin (20 pg), or Cerberus (1.6 ng) RNA and then treated with 50, 100, or 200 µM of SB43 and/or 20 µM DM. (A,B) Treatment with 100 µM SB43 + 20 µM DM (SB43+DM) mimics Noggin's ability to suppress pSmad1/5/8 and pSmad2, as determined by western blot. (B) Suppression of both pSmad1/5/8 and pSmad2 is only complete with treatment of both SB43+DM. Smad1, Smad2, and β-actin served as loading controls. (C) RT-PCR analysis of animal caps shows that Noggin or SB43+DM±Smad2 induces expression of rax, pax6, six3, and otx2, while repressing tbx3. Histone H4 (h4) was used as a loading control. (D–H) DM+SB43±Smad2 treatment mimics the retinal integration efficiency of Noggin. (D) Smad2-injected cells treated with DMSO remain in the skin, while (E) Nog+Smad2 injected cells form retina in all animals. (F) SB43+DM treatment alone and (G) with Smad2 direct pluripotent cells to the retina. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (H) Quantification of ACT assay results shows the synergistic effect of adding DM and SB43 to generate retina (N = 3). Green, YFP; red, rod photoreceptor marker, XAP2; blue, DAPI staining. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
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f05: Dual inhibition of Smad1/5/8 and Smad2 activity with chemical inhibitors DM and SB43 is sufficient to drive retinal formation.Embryos were injected with Smad2 (50 pg), Noggin (20 pg), or Cerberus (1.6 ng) RNA and then treated with 50, 100, or 200 µM of SB43 and/or 20 µM DM. (A,B) Treatment with 100 µM SB43 + 20 µM DM (SB43+DM) mimics Noggin's ability to suppress pSmad1/5/8 and pSmad2, as determined by western blot. (B) Suppression of both pSmad1/5/8 and pSmad2 is only complete with treatment of both SB43+DM. Smad1, Smad2, and β-actin served as loading controls. (C) RT-PCR analysis of animal caps shows that Noggin or SB43+DM±Smad2 induces expression of rax, pax6, six3, and otx2, while repressing tbx3. Histone H4 (h4) was used as a loading control. (D–H) DM+SB43±Smad2 treatment mimics the retinal integration efficiency of Noggin. (D) Smad2-injected cells treated with DMSO remain in the skin, while (E) Nog+Smad2 injected cells form retina in all animals. (F) SB43+DM treatment alone and (G) with Smad2 direct pluripotent cells to the retina. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (H) Quantification of ACT assay results shows the synergistic effect of adding DM and SB43 to generate retina (N = 3). Green, YFP; red, rod photoreceptor marker, XAP2; blue, DAPI staining. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.

Mentions: If complete inhibition of both pathways is required for efficient retina formation in pluripotent cells, we would expect blocking both Smad1 and 2 phosphorylation by chemical inhibitors would produce retinal cells in the pluripotent tissue. Again, DM was used to inhibit pSmad1/5/8, while the small molecule inhibitor, SB431542 (SB43), was used to inhibit pSmad2 (Inman et al., 2002; Laping et al., 2002). We first determined the optimal concentration of each chemical inhibitor to selectively inhibit each pathway. First, treating animal caps with SB43 significantly repressed pSmad2, with only a slight reduction of pSmad1/5/8 at 50 µM (99.5±15.3%; Fig. 5A, lane 5) and 100 µM (75.3±25.4%; Fig. 5A, lane 6), whereas treatment with 200 µM SB43 significantly reduced pSmad1/5/8 (39±27%; Fig. 5A, lane 7). Consistent with previously published data, we found that 30 µM lowered Smad2 activity, so we used dorsomorphin at 20 µM, (Fig. 5B, lane 3; Yu et al., 2008). Treatment with 100 µM SB43 and 20 µM DM (SB43+DM) was sufficient to repress both pSmad1/5/8 and pSmad2 (3.0±1.7%, 9.7±1.3%, respectively; Fig. 5A, lane 8) as effectively as injection with Noggin (2.7±2.2%, 7.0±4.0%; Fig. 5A, compare lanes 3,8).


Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells.

Wong KA, Trembley M, Abd Wahab S, Viczian AS - Biol Open (2015)

Dual inhibition of Smad1/5/8 and Smad2 activity with chemical inhibitors DM and SB43 is sufficient to drive retinal formation.Embryos were injected with Smad2 (50 pg), Noggin (20 pg), or Cerberus (1.6 ng) RNA and then treated with 50, 100, or 200 µM of SB43 and/or 20 µM DM. (A,B) Treatment with 100 µM SB43 + 20 µM DM (SB43+DM) mimics Noggin's ability to suppress pSmad1/5/8 and pSmad2, as determined by western blot. (B) Suppression of both pSmad1/5/8 and pSmad2 is only complete with treatment of both SB43+DM. Smad1, Smad2, and β-actin served as loading controls. (C) RT-PCR analysis of animal caps shows that Noggin or SB43+DM±Smad2 induces expression of rax, pax6, six3, and otx2, while repressing tbx3. Histone H4 (h4) was used as a loading control. (D–H) DM+SB43±Smad2 treatment mimics the retinal integration efficiency of Noggin. (D) Smad2-injected cells treated with DMSO remain in the skin, while (E) Nog+Smad2 injected cells form retina in all animals. (F) SB43+DM treatment alone and (G) with Smad2 direct pluripotent cells to the retina. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (H) Quantification of ACT assay results shows the synergistic effect of adding DM and SB43 to generate retina (N = 3). Green, YFP; red, rod photoreceptor marker, XAP2; blue, DAPI staining. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
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f05: Dual inhibition of Smad1/5/8 and Smad2 activity with chemical inhibitors DM and SB43 is sufficient to drive retinal formation.Embryos were injected with Smad2 (50 pg), Noggin (20 pg), or Cerberus (1.6 ng) RNA and then treated with 50, 100, or 200 µM of SB43 and/or 20 µM DM. (A,B) Treatment with 100 µM SB43 + 20 µM DM (SB43+DM) mimics Noggin's ability to suppress pSmad1/5/8 and pSmad2, as determined by western blot. (B) Suppression of both pSmad1/5/8 and pSmad2 is only complete with treatment of both SB43+DM. Smad1, Smad2, and β-actin served as loading controls. (C) RT-PCR analysis of animal caps shows that Noggin or SB43+DM±Smad2 induces expression of rax, pax6, six3, and otx2, while repressing tbx3. Histone H4 (h4) was used as a loading control. (D–H) DM+SB43±Smad2 treatment mimics the retinal integration efficiency of Noggin. (D) Smad2-injected cells treated with DMSO remain in the skin, while (E) Nog+Smad2 injected cells form retina in all animals. (F) SB43+DM treatment alone and (G) with Smad2 direct pluripotent cells to the retina. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (H) Quantification of ACT assay results shows the synergistic effect of adding DM and SB43 to generate retina (N = 3). Green, YFP; red, rod photoreceptor marker, XAP2; blue, DAPI staining. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
Mentions: If complete inhibition of both pathways is required for efficient retina formation in pluripotent cells, we would expect blocking both Smad1 and 2 phosphorylation by chemical inhibitors would produce retinal cells in the pluripotent tissue. Again, DM was used to inhibit pSmad1/5/8, while the small molecule inhibitor, SB431542 (SB43), was used to inhibit pSmad2 (Inman et al., 2002; Laping et al., 2002). We first determined the optimal concentration of each chemical inhibitor to selectively inhibit each pathway. First, treating animal caps with SB43 significantly repressed pSmad2, with only a slight reduction of pSmad1/5/8 at 50 µM (99.5±15.3%; Fig. 5A, lane 5) and 100 µM (75.3±25.4%; Fig. 5A, lane 6), whereas treatment with 200 µM SB43 significantly reduced pSmad1/5/8 (39±27%; Fig. 5A, lane 7). Consistent with previously published data, we found that 30 µM lowered Smad2 activity, so we used dorsomorphin at 20 µM, (Fig. 5B, lane 3; Yu et al., 2008). Treatment with 100 µM SB43 and 20 µM DM (SB43+DM) was sufficient to repress both pSmad1/5/8 and pSmad2 (3.0±1.7%, 9.7±1.3%, respectively; Fig. 5A, lane 8) as effectively as injection with Noggin (2.7±2.2%, 7.0±4.0%; Fig. 5A, compare lanes 3,8).

Bottom Line: We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos.We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone.In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA The Center for Vision Research, SUNY Eye Institute, Upstate Medical University, Syracuse, NY 13210, USA.

No MeSH data available.


Related in: MedlinePlus