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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

Injection of Smad1-AVA (S1-AVA) and Smad2-P445H (S2-P445H) act additively to cause expansion of eye field.Whole mount in situ hybridization for the eye field marker, rax (A–E) conducted on stage 15 embryos unilaterally injected with 125 pg of S1-AVA RNA, 3 ng of S2-P445H and 100 pg of β-gal. Area of rax expression was calculated by measuring the region within the dashed yellow lines on each side of the midline (white dotted line) as shown. Graph shows the ratio of the area of the injected side to the uninjected side. Red β-gal stain indicates injected side. Scale bar, 500 µm. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
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f04: Injection of Smad1-AVA (S1-AVA) and Smad2-P445H (S2-P445H) act additively to cause expansion of eye field.Whole mount in situ hybridization for the eye field marker, rax (A–E) conducted on stage 15 embryos unilaterally injected with 125 pg of S1-AVA RNA, 3 ng of S2-P445H and 100 pg of β-gal. Area of rax expression was calculated by measuring the region within the dashed yellow lines on each side of the midline (white dotted line) as shown. Graph shows the ratio of the area of the injected side to the uninjected side. Red β-gal stain indicates injected side. Scale bar, 500 µm. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.

Mentions: To better understand the role of BMP and Activin inhibition on retinal development in vivo, we attempted to block these pathways in whole embryos using two previously characterized dominant-negative Smad mutants. To block BMP signals, we injected a dominant-negative Smad1 mutant (Smad1-AVA) in which the C-terminal phosphorylated serines (Ser-463/465) were mutated to alanines to prevent phosphorylation but maintain receptor and co-factor interactions (Nojima et al., 2010). To block Activin signals, we injected a dominant-negative Smad2 mutant (Smad2-P445H), which harbors a point mutation in the MH2 domain that alters the structure and prevents C-terminal phosphorylation (Eppert et al., 1996). Mutants were unilaterally injected with RNA, including β-gal RNA as a tracer. Neural plate expansion was measured by in situ hybridization for the eye field marker, rax. The rax expression pattern remained bilaterally symmetric with injection of β-gal (Fig. 4A; injected:uninjected ratio of 1.05±0.01, n = 67). If BMP and Activin repression are both necessary for efficient eye formation, we would expect to see an expansion of the eye field when both mutants are expressed. Indeed, we saw that when injected alone, Smad1-AVA and Smad2-P445H slightly expanded rax expression domains (Fig. 4C; 1.18±0.02, n = 54; Fig. 4D; 1.18±0.03, n = 52, respectively). Eye field expansion was further increased by the injection of both Smad1-AVA and Smad2-P445H together (Fig. 4E; 1.30±0.03, n = 54). This suggests that, in the embryo, reducing Smad2 activity enhances the effect of reducing Smad1 activity to expand rax expression. However, this expansion was not as great as injecting Noggin alone (Fig. 4B; 1.69±0.07, n = 57). It is known that these constructs fail to completely abolish endogenous Smad1 or Smad2 activity (Nojima et al., 2010; Eppert et al., 1996; Hoodless et al., 1999; Prunier et al., 2001). This would explain the difference we observed between rax expansion in Noggin samples versus those treated with the dominant-negative Smads.


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)

Injection of Smad1-AVA (S1-AVA) and Smad2-P445H (S2-P445H) act additively to cause expansion of eye field.Whole mount in situ hybridization for the eye field marker, rax (A–E) conducted on stage 15 embryos unilaterally injected with 125 pg of S1-AVA RNA, 3 ng of S2-P445H and 100 pg of β-gal. Area of rax expression was calculated by measuring the region within the dashed yellow lines on each side of the midline (white dotted line) as shown. Graph shows the ratio of the area of the injected side to the uninjected side. Red β-gal stain indicates injected side. Scale bar, 500 µm. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400599&req=5

f04: Injection of Smad1-AVA (S1-AVA) and Smad2-P445H (S2-P445H) act additively to cause expansion of eye field.Whole mount in situ hybridization for the eye field marker, rax (A–E) conducted on stage 15 embryos unilaterally injected with 125 pg of S1-AVA RNA, 3 ng of S2-P445H and 100 pg of β-gal. Area of rax expression was calculated by measuring the region within the dashed yellow lines on each side of the midline (white dotted line) as shown. Graph shows the ratio of the area of the injected side to the uninjected side. Red β-gal stain indicates injected side. Scale bar, 500 µm. Error bars  =  ±s.e.m.; **p<0.01; ***p<0.001.
Mentions: To better understand the role of BMP and Activin inhibition on retinal development in vivo, we attempted to block these pathways in whole embryos using two previously characterized dominant-negative Smad mutants. To block BMP signals, we injected a dominant-negative Smad1 mutant (Smad1-AVA) in which the C-terminal phosphorylated serines (Ser-463/465) were mutated to alanines to prevent phosphorylation but maintain receptor and co-factor interactions (Nojima et al., 2010). To block Activin signals, we injected a dominant-negative Smad2 mutant (Smad2-P445H), which harbors a point mutation in the MH2 domain that alters the structure and prevents C-terminal phosphorylation (Eppert et al., 1996). Mutants were unilaterally injected with RNA, including β-gal RNA as a tracer. Neural plate expansion was measured by in situ hybridization for the eye field marker, rax. The rax expression pattern remained bilaterally symmetric with injection of β-gal (Fig. 4A; injected:uninjected ratio of 1.05±0.01, n = 67). If BMP and Activin repression are both necessary for efficient eye formation, we would expect to see an expansion of the eye field when both mutants are expressed. Indeed, we saw that when injected alone, Smad1-AVA and Smad2-P445H slightly expanded rax expression domains (Fig. 4C; 1.18±0.02, n = 54; Fig. 4D; 1.18±0.03, n = 52, respectively). Eye field expansion was further increased by the injection of both Smad1-AVA and Smad2-P445H together (Fig. 4E; 1.30±0.03, n = 54). This suggests that, in the embryo, reducing Smad2 activity enhances the effect of reducing Smad1 activity to expand rax expression. However, this expansion was not as great as injecting Noggin alone (Fig. 4B; 1.69±0.07, n = 57). It is known that these constructs fail to completely abolish endogenous Smad1 or Smad2 activity (Nojima et al., 2010; Eppert et al., 1996; Hoodless et al., 1999; Prunier et al., 2001). This would explain the difference we observed between rax expansion in Noggin samples versus those treated with the dominant-negative Smads.

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