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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

DM and SB43 treatment results in eye field expansion.(A–E) In situ hybridization for the eye field marker, rax, in embryos treated with DMSO (A), DM (B,C) or DM+SB43 (D,E) from stage 9–15. Eye field expansion was determined by measuring the area of rax expression (dashed yellow line) normalized to the area of the dorsal face of the embryo (dashed white line), as shown in the graph. Error bars  =  ±s.e.m.; ***p<0.001. Dotted line on graph separates single treatment from dual treatment. Scale bar, 500 µm; n = number of embryos.
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f06: DM and SB43 treatment results in eye field expansion.(A–E) In situ hybridization for the eye field marker, rax, in embryos treated with DMSO (A), DM (B,C) or DM+SB43 (D,E) from stage 9–15. Eye field expansion was determined by measuring the area of rax expression (dashed yellow line) normalized to the area of the dorsal face of the embryo (dashed white line), as shown in the graph. Error bars  =  ±s.e.m.; ***p<0.001. Dotted line on graph separates single treatment from dual treatment. Scale bar, 500 µm; n = number of embryos.

Mentions: The above results suggest that BMP and Activin inhibition is sufficient to direct isolated pluripotent cells to generate retina. We were next interested if the same phenomenon was true in vivo. We treated embryos with varying concentrations of DM and/or SB43, and probed for rax expression by whole mount in situ hybridization. We hypothesized that if treatment with DM and SB43 is sufficient to drive retina formation in vivo, then the rax expression domain would be expanded. We observed that embryos treated with 10 µM DM resulted in an insignificant expansion of the eye field compared to DMSO treated embryos (Fig. 6A,B; DMSO, 9.8±0.3% of the anterior embryo face; DM 10 µM, 10.8±0.2%). We first see expansion of the rax expression domains with treatment of 20 µM DM (Fig. 6C, 11.4±0.3%). This expansion was further increased by treatment with both 20 µM DM and 100 µM SB43, as previously used in our transplant assays (Fig. 6E, 13.5±0.3%). Treatment with a more dilute SB43+DM treatment (10 µM DM and 50 µM SB43) resulted in no significant expansion (Fig. 6D, 10.8±0.2%). Thus, inhibiting BMP and Activin signaling using these two chemical inhibitors can also promote eye field expansion in vivo.


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)

DM and SB43 treatment results in eye field expansion.(A–E) In situ hybridization for the eye field marker, rax, in embryos treated with DMSO (A), DM (B,C) or DM+SB43 (D,E) from stage 9–15. Eye field expansion was determined by measuring the area of rax expression (dashed yellow line) normalized to the area of the dorsal face of the embryo (dashed white line), as shown in the graph. Error bars  =  ±s.e.m.; ***p<0.001. Dotted line on graph separates single treatment from dual treatment. Scale bar, 500 µm; n = number of embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f06: DM and SB43 treatment results in eye field expansion.(A–E) In situ hybridization for the eye field marker, rax, in embryos treated with DMSO (A), DM (B,C) or DM+SB43 (D,E) from stage 9–15. Eye field expansion was determined by measuring the area of rax expression (dashed yellow line) normalized to the area of the dorsal face of the embryo (dashed white line), as shown in the graph. Error bars  =  ±s.e.m.; ***p<0.001. Dotted line on graph separates single treatment from dual treatment. Scale bar, 500 µm; n = number of embryos.
Mentions: The above results suggest that BMP and Activin inhibition is sufficient to direct isolated pluripotent cells to generate retina. We were next interested if the same phenomenon was true in vivo. We treated embryos with varying concentrations of DM and/or SB43, and probed for rax expression by whole mount in situ hybridization. We hypothesized that if treatment with DM and SB43 is sufficient to drive retina formation in vivo, then the rax expression domain would be expanded. We observed that embryos treated with 10 µM DM resulted in an insignificant expansion of the eye field compared to DMSO treated embryos (Fig. 6A,B; DMSO, 9.8±0.3% of the anterior embryo face; DM 10 µM, 10.8±0.2%). We first see expansion of the rax expression domains with treatment of 20 µM DM (Fig. 6C, 11.4±0.3%). This expansion was further increased by treatment with both 20 µM DM and 100 µM SB43, as previously used in our transplant assays (Fig. 6E, 13.5±0.3%). Treatment with a more dilute SB43+DM treatment (10 µM DM and 50 µM SB43) resulted in no significant expansion (Fig. 6D, 10.8±0.2%). Thus, inhibiting BMP and Activin signaling using these two chemical inhibitors can also promote eye field expansion in vivo.

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