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In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish.

Prykhozhij SV - PLoS ONE (2010)

Bottom Line: While in shh(-/-) mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh(-/-) mutants.Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53(-/-)shh(-/-) mutant retina suggesting the effect of p53 on retinal differentiation.Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. rykhozh@embl.de

ABSTRACT

Background: Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/principal findings: Analysis of the zebrafish shh(-/-) mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh(-/-) mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh(-/-) mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh(-/-) mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53(-/-)shh(-/-) mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions: Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.

Show MeSH
Hh signaling activation by dnPKA-GFP decreases p53 target cyclinG1 expression and suppresses apoptosis in shh−/− mutant embryos.Embryos from shh+/– parent fish were injected with either dnPKA-GFP or EGFP mRNA, grown to 12-somite stage and in situ stained for patched1, target gene of Hh signaling, and cyclinG1, p53 target gene, and ApopTag staining was performed to characterize the level of apoptosis. (A) Injection of dnPKA-GFP led to a strongly increased and more widely spread expression of patched1 (category “high”) in 87,7+/−3,8% of injected embryos, the rest having normal wild-type or low shh−/− mutant patched1 expression comparable to expression of patched1 in EGFP-injected embryos from shh+/– parent fish (category “low”). (B) Expression of cyclinG1 in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate expression levels in shh−/− mutant and wild-type embryos, respectively. (C) ApopTag staining of apoptotic cells in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate levels of apoptosis in shh−/− mutant and wild-type embryos, respectively. (D) Statistical analysis of results from injections of dnPKA-GFP and EGFP mRNA into embryos from shh+/– parent fish. All of EGFP-injected embryos had “low” expression of patched1, 25,3 +/− 1,15% (Mean +/− standard deviation) of them had “high” expression of cyclinG1 and 24,7 +/− 1,53% had “high” apoptosis levels. dnPKA-GFP mRNA injection led to “high” expression of patched1 in 87,7 +/− 3,8% of embryos and decreased expression of cyclinG1 and apoptosis levels: “high” proportions are 6 +/− 2% and 5,8 +/− 2,4%, respectively. The experiment was done 3 times and 60 embryos were used for each staining. Asterisks (***) on top of shh−/− mutant bars indicate significant statistical differences of proportions (Fisher's exact test, P-value<0,001).
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pone-0013549-g006: Hh signaling activation by dnPKA-GFP decreases p53 target cyclinG1 expression and suppresses apoptosis in shh−/− mutant embryos.Embryos from shh+/– parent fish were injected with either dnPKA-GFP or EGFP mRNA, grown to 12-somite stage and in situ stained for patched1, target gene of Hh signaling, and cyclinG1, p53 target gene, and ApopTag staining was performed to characterize the level of apoptosis. (A) Injection of dnPKA-GFP led to a strongly increased and more widely spread expression of patched1 (category “high”) in 87,7+/−3,8% of injected embryos, the rest having normal wild-type or low shh−/− mutant patched1 expression comparable to expression of patched1 in EGFP-injected embryos from shh+/– parent fish (category “low”). (B) Expression of cyclinG1 in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate expression levels in shh−/− mutant and wild-type embryos, respectively. (C) ApopTag staining of apoptotic cells in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate levels of apoptosis in shh−/− mutant and wild-type embryos, respectively. (D) Statistical analysis of results from injections of dnPKA-GFP and EGFP mRNA into embryos from shh+/– parent fish. All of EGFP-injected embryos had “low” expression of patched1, 25,3 +/− 1,15% (Mean +/− standard deviation) of them had “high” expression of cyclinG1 and 24,7 +/− 1,53% had “high” apoptosis levels. dnPKA-GFP mRNA injection led to “high” expression of patched1 in 87,7 +/− 3,8% of embryos and decreased expression of cyclinG1 and apoptosis levels: “high” proportions are 6 +/− 2% and 5,8 +/− 2,4%, respectively. The experiment was done 3 times and 60 embryos were used for each staining. Asterisks (***) on top of shh−/− mutant bars indicate significant statistical differences of proportions (Fisher's exact test, P-value<0,001).

Mentions: In tissue culture experiments, p53 activation has been shown to be inhibited by Gli-mediated activation of an unknown p53 pathway inhibitor [18]. This leads to the question whether activation of Hh signalling inhibits p53 pathway in vivo in the absence of Shh. To activate the canonical Hh/Gli signaling pathway, I injected mRNA of dominant-negative PKA fused with GFP (dnPKA-GFP) [50], [51] or EGFP mRNA as a control into progeny of fish heterozygous for the shh deletion. Given the limited mRNA stability, I analysed the effects of RNA injections at the 12 somites stage, when shh−/− mutant embryos already show a higher level of p53 target gene expression and apoptosis than wild-type embryos (Fig. S2). While all the EGFP-injected embryos showed unaltered wild-type or shh−/− mutant patched1 expression pattern (Fig. 6A, D, “low”), over-expression of dnPKA-GFP led to ectopic spreading of patched1 expression in 87,7 +/− 3,8% of embryos (Fig. 6A, D, “high”). I next assessed expression of cyclinG1, p53 target gene, after mRNA injections by visually scoring expression strength as “high” or “low” based on staining of uninjected embryos (Fig. 6B), where embryos with “high” cyclinG1 expression were inferred to be shh−/− mutant and those with “low” expression were wild-type (control stainings of wild-type embryos, not shown). In this experiment, the effect of dnPKA-GFP on p53 target gene expression is inferred from the change in proportion of embryos with stronger cyclinG1 expression. While after EGFP mRNA injection the proportion of embryos with “high” expression of cyclinG1 was 25,3 +/− 1,2% (Fig. 6D), only 6 +/− 2% (Fig. 6D) of dnPKA-GFP -injected embryos had perceptibly stronger cyclinG1 expression than the other embryos in the samples (Fig. 6D). Overexpression of dnPKA-GFP also reduced the proportion of embryos with higher apoptosis levels (Fig. 6C) to 5,8 +/− 2,4% (Fig. 6D) (Fig. 6D) compared to 24,7 +/− 1,53% (Fig. 6D) among EGFP-injected embryos (Fig. 6D). Taken together, these data indicate that activation of Hh signalling using dnPKA-GFP can suppress p53 target expression and apoptosis.


In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish.

Prykhozhij SV - PLoS ONE (2010)

Hh signaling activation by dnPKA-GFP decreases p53 target cyclinG1 expression and suppresses apoptosis in shh−/− mutant embryos.Embryos from shh+/– parent fish were injected with either dnPKA-GFP or EGFP mRNA, grown to 12-somite stage and in situ stained for patched1, target gene of Hh signaling, and cyclinG1, p53 target gene, and ApopTag staining was performed to characterize the level of apoptosis. (A) Injection of dnPKA-GFP led to a strongly increased and more widely spread expression of patched1 (category “high”) in 87,7+/−3,8% of injected embryos, the rest having normal wild-type or low shh−/− mutant patched1 expression comparable to expression of patched1 in EGFP-injected embryos from shh+/– parent fish (category “low”). (B) Expression of cyclinG1 in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate expression levels in shh−/− mutant and wild-type embryos, respectively. (C) ApopTag staining of apoptotic cells in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate levels of apoptosis in shh−/− mutant and wild-type embryos, respectively. (D) Statistical analysis of results from injections of dnPKA-GFP and EGFP mRNA into embryos from shh+/– parent fish. All of EGFP-injected embryos had “low” expression of patched1, 25,3 +/− 1,15% (Mean +/− standard deviation) of them had “high” expression of cyclinG1 and 24,7 +/− 1,53% had “high” apoptosis levels. dnPKA-GFP mRNA injection led to “high” expression of patched1 in 87,7 +/− 3,8% of embryos and decreased expression of cyclinG1 and apoptosis levels: “high” proportions are 6 +/− 2% and 5,8 +/− 2,4%, respectively. The experiment was done 3 times and 60 embryos were used for each staining. Asterisks (***) on top of shh−/− mutant bars indicate significant statistical differences of proportions (Fisher's exact test, P-value<0,001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958845&req=5

pone-0013549-g006: Hh signaling activation by dnPKA-GFP decreases p53 target cyclinG1 expression and suppresses apoptosis in shh−/− mutant embryos.Embryos from shh+/– parent fish were injected with either dnPKA-GFP or EGFP mRNA, grown to 12-somite stage and in situ stained for patched1, target gene of Hh signaling, and cyclinG1, p53 target gene, and ApopTag staining was performed to characterize the level of apoptosis. (A) Injection of dnPKA-GFP led to a strongly increased and more widely spread expression of patched1 (category “high”) in 87,7+/−3,8% of injected embryos, the rest having normal wild-type or low shh−/− mutant patched1 expression comparable to expression of patched1 in EGFP-injected embryos from shh+/– parent fish (category “low”). (B) Expression of cyclinG1 in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate expression levels in shh−/− mutant and wild-type embryos, respectively. (C) ApopTag staining of apoptotic cells in uninjected embryos from shh+/– parent fish. Categories “high” and “low” indicate levels of apoptosis in shh−/− mutant and wild-type embryos, respectively. (D) Statistical analysis of results from injections of dnPKA-GFP and EGFP mRNA into embryos from shh+/– parent fish. All of EGFP-injected embryos had “low” expression of patched1, 25,3 +/− 1,15% (Mean +/− standard deviation) of them had “high” expression of cyclinG1 and 24,7 +/− 1,53% had “high” apoptosis levels. dnPKA-GFP mRNA injection led to “high” expression of patched1 in 87,7 +/− 3,8% of embryos and decreased expression of cyclinG1 and apoptosis levels: “high” proportions are 6 +/− 2% and 5,8 +/− 2,4%, respectively. The experiment was done 3 times and 60 embryos were used for each staining. Asterisks (***) on top of shh−/− mutant bars indicate significant statistical differences of proportions (Fisher's exact test, P-value<0,001).
Mentions: In tissue culture experiments, p53 activation has been shown to be inhibited by Gli-mediated activation of an unknown p53 pathway inhibitor [18]. This leads to the question whether activation of Hh signalling inhibits p53 pathway in vivo in the absence of Shh. To activate the canonical Hh/Gli signaling pathway, I injected mRNA of dominant-negative PKA fused with GFP (dnPKA-GFP) [50], [51] or EGFP mRNA as a control into progeny of fish heterozygous for the shh deletion. Given the limited mRNA stability, I analysed the effects of RNA injections at the 12 somites stage, when shh−/− mutant embryos already show a higher level of p53 target gene expression and apoptosis than wild-type embryos (Fig. S2). While all the EGFP-injected embryos showed unaltered wild-type or shh−/− mutant patched1 expression pattern (Fig. 6A, D, “low”), over-expression of dnPKA-GFP led to ectopic spreading of patched1 expression in 87,7 +/− 3,8% of embryos (Fig. 6A, D, “high”). I next assessed expression of cyclinG1, p53 target gene, after mRNA injections by visually scoring expression strength as “high” or “low” based on staining of uninjected embryos (Fig. 6B), where embryos with “high” cyclinG1 expression were inferred to be shh−/− mutant and those with “low” expression were wild-type (control stainings of wild-type embryos, not shown). In this experiment, the effect of dnPKA-GFP on p53 target gene expression is inferred from the change in proportion of embryos with stronger cyclinG1 expression. While after EGFP mRNA injection the proportion of embryos with “high” expression of cyclinG1 was 25,3 +/− 1,2% (Fig. 6D), only 6 +/− 2% (Fig. 6D) of dnPKA-GFP -injected embryos had perceptibly stronger cyclinG1 expression than the other embryos in the samples (Fig. 6D). Overexpression of dnPKA-GFP also reduced the proportion of embryos with higher apoptosis levels (Fig. 6C) to 5,8 +/− 2,4% (Fig. 6D) (Fig. 6D) compared to 24,7 +/− 1,53% (Fig. 6D) among EGFP-injected embryos (Fig. 6D). Taken together, these data indicate that activation of Hh signalling using dnPKA-GFP can suppress p53 target expression and apoptosis.

Bottom Line: While in shh(-/-) mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh(-/-) mutants.Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53(-/-)shh(-/-) mutant retina suggesting the effect of p53 on retinal differentiation.Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. rykhozh@embl.de

ABSTRACT

Background: Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/principal findings: Analysis of the zebrafish shh(-/-) mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh(-/-) mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh(-/-) mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh(-/-) mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53(-/-)shh(-/-) mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions: Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.

Show MeSH