Limits...
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

Noggin inhibits Smad1/5/8 and Smad2 phosphorylation in a concentration-dependent manner.(A) Western blot of animal caps isolated from embryos injected with specified amount of Noggin RNA with and without 50 pg Smad2 RNA. Smad1, Smad2, and β-actin served as loading controls. (B) Densitometric analysis of western blots shows that higher concentrations of Noggin inhibit pSmad1/5/8 and pSmad2 (N = 3). (C) Noggin inhibits BMP and Activin pathway-specific gene transcription. Noggin-treated caps can inhibit the epithelia marker, xk81, and mesoderm marker, xbra, as determined through RT-PCR. Conversely, DM affects BMP, not Activin, pathway gene transcripts since it can only affect xk81 expression; N = 3. Error bars  =  ±s.e.m.; *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400599&req=5

f02: Noggin inhibits Smad1/5/8 and Smad2 phosphorylation in a concentration-dependent manner.(A) Western blot of animal caps isolated from embryos injected with specified amount of Noggin RNA with and without 50 pg Smad2 RNA. Smad1, Smad2, and β-actin served as loading controls. (B) Densitometric analysis of western blots shows that higher concentrations of Noggin inhibit pSmad1/5/8 and pSmad2 (N = 3). (C) Noggin inhibits BMP and Activin pathway-specific gene transcription. Noggin-treated caps can inhibit the epithelia marker, xk81, and mesoderm marker, xbra, as determined through RT-PCR. Conversely, DM affects BMP, not Activin, pathway gene transcripts since it can only affect xk81 expression; N = 3. Error bars  =  ±s.e.m.; *p<0.05.

Mentions: Activin signaling (via Smad2 phosphorylation) can be stimulated in animal cap cells as early as stage 6 up to stage 11 by adding the Activin ligand (Grimm and Gurdon, 2002). Adding Activin to animal cap cells has allowed closer examination of Smad2 activity in this promiscuous tissue (Chang et al., 1997). Since we were interested in the intrinsic changes regulated by Noggin, we injected a small amount of Smad2 mRNA (50 pg) into embryos, in order to more easily visualize the effect of Noggin on Smad2 activity. While this concentration was high enough to detect pSmad2, it was low enough to prevent animal cap elongation, which is phenotypical of sustained Activin signaling (Asashima et al., 1990; Smith et al., 1990; Thomsen et al., 1990). Consistent with our investigation of endogenous Activin signaling, we found that Noggin could reduce both pSmad1/5/8 and pSmad2 (Fig. 2A). Smad1 and Smad2 protein levels remained constant, suggesting that Smad degradation pathways were not responsible for this change in activity. Although the decrease in Smad2 activity was statistically significant at all Noggin concentrations tested, pSmad2 was repressed more completely when 5 and 20 pg Noggin RNA was injected (Fig. 2B).


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)

Noggin inhibits Smad1/5/8 and Smad2 phosphorylation in a concentration-dependent manner.(A) Western blot of animal caps isolated from embryos injected with specified amount of Noggin RNA with and without 50 pg Smad2 RNA. Smad1, Smad2, and β-actin served as loading controls. (B) Densitometric analysis of western blots shows that higher concentrations of Noggin inhibit pSmad1/5/8 and pSmad2 (N = 3). (C) Noggin inhibits BMP and Activin pathway-specific gene transcription. Noggin-treated caps can inhibit the epithelia marker, xk81, and mesoderm marker, xbra, as determined through RT-PCR. Conversely, DM affects BMP, not Activin, pathway gene transcripts since it can only affect xk81 expression; N = 3. Error bars  =  ±s.e.m.; *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f02: Noggin inhibits Smad1/5/8 and Smad2 phosphorylation in a concentration-dependent manner.(A) Western blot of animal caps isolated from embryos injected with specified amount of Noggin RNA with and without 50 pg Smad2 RNA. Smad1, Smad2, and β-actin served as loading controls. (B) Densitometric analysis of western blots shows that higher concentrations of Noggin inhibit pSmad1/5/8 and pSmad2 (N = 3). (C) Noggin inhibits BMP and Activin pathway-specific gene transcription. Noggin-treated caps can inhibit the epithelia marker, xk81, and mesoderm marker, xbra, as determined through RT-PCR. Conversely, DM affects BMP, not Activin, pathway gene transcripts since it can only affect xk81 expression; N = 3. Error bars  =  ±s.e.m.; *p<0.05.
Mentions: Activin signaling (via Smad2 phosphorylation) can be stimulated in animal cap cells as early as stage 6 up to stage 11 by adding the Activin ligand (Grimm and Gurdon, 2002). Adding Activin to animal cap cells has allowed closer examination of Smad2 activity in this promiscuous tissue (Chang et al., 1997). Since we were interested in the intrinsic changes regulated by Noggin, we injected a small amount of Smad2 mRNA (50 pg) into embryos, in order to more easily visualize the effect of Noggin on Smad2 activity. While this concentration was high enough to detect pSmad2, it was low enough to prevent animal cap elongation, which is phenotypical of sustained Activin signaling (Asashima et al., 1990; Smith et al., 1990; Thomsen et al., 1990). Consistent with our investigation of endogenous Activin signaling, we found that Noggin could reduce both pSmad1/5/8 and pSmad2 (Fig. 2A). Smad1 and Smad2 protein levels remained constant, suggesting that Smad degradation pathways were not responsible for this change in activity. Although the decrease in Smad2 activity was statistically significant at all Noggin concentrations tested, pSmad2 was repressed more completely when 5 and 20 pg Noggin RNA was injected (Fig. 2B).

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