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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

(A-D) Whole mount in situ hybridization for BMP (A,B) and Activin (C,D) type II receptors in stage 15 embryos show expression in the eye field (yellow), outlined with the dashed lines. (A) and (C) show the front view, while (B) and (D) show a side view. (E) Expression of truncated BMP (tBRII, 500 pg) or Activin (ΔXAR1, 1 ng) receptors individually suppress pSmad1/5/8, but signaling is repressed further with expression of both tBRII+ΔXAR1. (F) pSmad2 is also repressed with the expression of both tBRII and ΔXAR1. (G-K) Using the ACT assay, the tBRII+ΔXAR1-expressing cells end up in the retina more frequently than either the tBR or ΔXAR1-expressing pluripotent cells. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (K) ACT results quantified and statistics determined using a student's t-test, N=2. Error bars = ±s.e.m.; *p,0.05. Green, YFP; blue, DAPI staining. Dorsal ‘D’, ventral ‘V’, posterior ‘P’, anterior ‘A’.
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f03: (A-D) Whole mount in situ hybridization for BMP (A,B) and Activin (C,D) type II receptors in stage 15 embryos show expression in the eye field (yellow), outlined with the dashed lines. (A) and (C) show the front view, while (B) and (D) show a side view. (E) Expression of truncated BMP (tBRII, 500 pg) or Activin (ΔXAR1, 1 ng) receptors individually suppress pSmad1/5/8, but signaling is repressed further with expression of both tBRII+ΔXAR1. (F) pSmad2 is also repressed with the expression of both tBRII and ΔXAR1. (G-K) Using the ACT assay, the tBRII+ΔXAR1-expressing cells end up in the retina more frequently than either the tBR or ΔXAR1-expressing pluripotent cells. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (K) ACT results quantified and statistics determined using a student's t-test, N=2. Error bars = ±s.e.m.; *p,0.05. Green, YFP; blue, DAPI staining. Dorsal ‘D’, ventral ‘V’, posterior ‘P’, anterior ‘A’.

Mentions: Both BMP4 and Activin signal through specific TGFβ receptors. If Noggin is acting upstream of both pathways, we expect that expression of dominant-negative receptors for both these pathways would mimic Noggin's ability to induce retina formation. We used two mutant membrane receptors, a truncated BMP type II receptor (tBRII) to preferentially block the BMP pathway, and a truncated Activin type II receptor (ΔXAR1) to preferentially block the Activin signaling pathway. Both mutants are able to bind endogenous ligands, but lack their C-terminal domains, preventing the phosphorylation of their complementary type I receptors and downstream signaling Smads (Hemmati-Brivanlou and Melton, 1992; Graff et al., 1994; Frisch and Wright, 1998). To confirm that these pathways have the potential to be activated, we performed in situ hybridization on xBMPrII (BMP receptor type II) and xStk2 (Activin A receptor type IIB precursor). We found that both receptors were expressed in the neural plate and eye field of stage 15 embryos (Fig. 3A–D), which is consistent with previous findings (Hemmati-Brivanlou and Melton, 1992). Also consistent with previous findings, we observed a significant decrease in pSmad1/5/8 with injection of tBRII compared to YFP-injected cells (46%; Fig. 3E, lane 3). Similarly, injection of ΔXAR1 significantly decreased pSmad2 (68%; Fig. 3F, lane 4; Hemmati-Brivanlou and Melton, 1992; Frisch and Wright, 1998). We also observed that injection of ΔXAR1 repressed pSmad1/5/8 (68%; Fig. 3E, lane 4), suggesting that ΔXAR1 can disrupt canonical BMP signaling, as others have reported (Wilson and Hemmati-Brivanlou, 1995; Frisch and Wright, 1998). We then wondered if tBRII disrupted Activin signaling. Indeed, we observed that injection of tBRII repressed pSmad2 (15%; Fig. 3F, lane 3) as effectively as injection of ΔXAR1 (17%; Fig. 3F, lane 4), suggesting that tBRII also represses both BMP and Activin signaling. When both are added together, we saw further reduction of both pSmad1/5/8 (12%) and pSmad2 (8%; lanes 5 in Fig. 3E,F), similar to levels observed when injecting cells with Noggin RNA (0%, pSmad1/5/8; 6%, pSmad2; lane 2 in Fig. 3E,F).


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)

(A-D) Whole mount in situ hybridization for BMP (A,B) and Activin (C,D) type II receptors in stage 15 embryos show expression in the eye field (yellow), outlined with the dashed lines. (A) and (C) show the front view, while (B) and (D) show a side view. (E) Expression of truncated BMP (tBRII, 500 pg) or Activin (ΔXAR1, 1 ng) receptors individually suppress pSmad1/5/8, but signaling is repressed further with expression of both tBRII+ΔXAR1. (F) pSmad2 is also repressed with the expression of both tBRII and ΔXAR1. (G-K) Using the ACT assay, the tBRII+ΔXAR1-expressing cells end up in the retina more frequently than either the tBR or ΔXAR1-expressing pluripotent cells. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (K) ACT results quantified and statistics determined using a student's t-test, N=2. Error bars = ±s.e.m.; *p,0.05. Green, YFP; blue, DAPI staining. Dorsal ‘D’, ventral ‘V’, posterior ‘P’, anterior ‘A’.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f03: (A-D) Whole mount in situ hybridization for BMP (A,B) and Activin (C,D) type II receptors in stage 15 embryos show expression in the eye field (yellow), outlined with the dashed lines. (A) and (C) show the front view, while (B) and (D) show a side view. (E) Expression of truncated BMP (tBRII, 500 pg) or Activin (ΔXAR1, 1 ng) receptors individually suppress pSmad1/5/8, but signaling is repressed further with expression of both tBRII+ΔXAR1. (F) pSmad2 is also repressed with the expression of both tBRII and ΔXAR1. (G-K) Using the ACT assay, the tBRII+ΔXAR1-expressing cells end up in the retina more frequently than either the tBR or ΔXAR1-expressing pluripotent cells. Scale bar, 50 µm. Dashed lines lie on outer and inner plexiform layers. (K) ACT results quantified and statistics determined using a student's t-test, N=2. Error bars = ±s.e.m.; *p,0.05. Green, YFP; blue, DAPI staining. Dorsal ‘D’, ventral ‘V’, posterior ‘P’, anterior ‘A’.
Mentions: Both BMP4 and Activin signal through specific TGFβ receptors. If Noggin is acting upstream of both pathways, we expect that expression of dominant-negative receptors for both these pathways would mimic Noggin's ability to induce retina formation. We used two mutant membrane receptors, a truncated BMP type II receptor (tBRII) to preferentially block the BMP pathway, and a truncated Activin type II receptor (ΔXAR1) to preferentially block the Activin signaling pathway. Both mutants are able to bind endogenous ligands, but lack their C-terminal domains, preventing the phosphorylation of their complementary type I receptors and downstream signaling Smads (Hemmati-Brivanlou and Melton, 1992; Graff et al., 1994; Frisch and Wright, 1998). To confirm that these pathways have the potential to be activated, we performed in situ hybridization on xBMPrII (BMP receptor type II) and xStk2 (Activin A receptor type IIB precursor). We found that both receptors were expressed in the neural plate and eye field of stage 15 embryos (Fig. 3A–D), which is consistent with previous findings (Hemmati-Brivanlou and Melton, 1992). Also consistent with previous findings, we observed a significant decrease in pSmad1/5/8 with injection of tBRII compared to YFP-injected cells (46%; Fig. 3E, lane 3). Similarly, injection of ΔXAR1 significantly decreased pSmad2 (68%; Fig. 3F, lane 4; Hemmati-Brivanlou and Melton, 1992; Frisch and Wright, 1998). We also observed that injection of ΔXAR1 repressed pSmad1/5/8 (68%; Fig. 3E, lane 4), suggesting that ΔXAR1 can disrupt canonical BMP signaling, as others have reported (Wilson and Hemmati-Brivanlou, 1995; Frisch and Wright, 1998). We then wondered if tBRII disrupted Activin signaling. Indeed, we observed that injection of tBRII repressed pSmad2 (15%; Fig. 3F, lane 3) as effectively as injection of ΔXAR1 (17%; Fig. 3F, lane 4), suggesting that tBRII also represses both BMP and Activin signaling. When both are added together, we saw further reduction of both pSmad1/5/8 (12%) and pSmad2 (8%; lanes 5 in Fig. 3E,F), similar to levels observed when injecting cells with Noggin RNA (0%, pSmad1/5/8; 6%, pSmad2; lane 2 in Fig. 3E,F).

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