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Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo.

Cavalieri V, Spinelli G - PLoS ONE (2015)

Bottom Line: Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae.Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae.Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Italy.

ABSTRACT
Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

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Related in: MedlinePlus

Rescue of nodal transcription by HD expression into TSA-treated embryos.(A) 0.1–0.2 pg of the hd mRNA, or a control out-of-frame strim1 transcript [53], were injected into zygotes exposed to TSA and total RNA isolated from the resulting embryos at the early blastula stage. (B) qPCR measurements of relative nodal transcript abundance in embryos exposed to TSA and injected with increasing amounts of the hd mRNA, compared to the nodal mRNA level of control unperturbed embryos. Data are normalized and indicated as in Fig 1C. (C) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd mRNA together with the TRCD red fluorescent tracer, and the phenotype of the resulting embryos was examined at the gastrula stage. (D) Representative examples of control gastrulae, TSA-treated larvae, and rescued embryos at the same stage injected with the hd mRNA, respectively ordered from left to right. Note that in both the rescued embryos, the progeny of the blastomere that received hd was embedded into the ventral side. (E) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd-gfp mRNA, and the resulting embryos at the early blastula stage were disaggregated into individual cells that were eventually segregated into two populations, based on GFP fluorescence, by way of FACS. (F) Representative example of embryos expressing HD-GFP clonally, observed at the early blastula stage, just before dissociation. (G) Changes in gene expression level of nodal assessed by qPCR in whole embryos exposed to TSA, and in subpopulations of cells sorted from rescued TSA-treated embryos clonally expressing HD-GFP. Data are normalized and indicated as in Fig 1C.
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pone.0143860.g004: Rescue of nodal transcription by HD expression into TSA-treated embryos.(A) 0.1–0.2 pg of the hd mRNA, or a control out-of-frame strim1 transcript [53], were injected into zygotes exposed to TSA and total RNA isolated from the resulting embryos at the early blastula stage. (B) qPCR measurements of relative nodal transcript abundance in embryos exposed to TSA and injected with increasing amounts of the hd mRNA, compared to the nodal mRNA level of control unperturbed embryos. Data are normalized and indicated as in Fig 1C. (C) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd mRNA together with the TRCD red fluorescent tracer, and the phenotype of the resulting embryos was examined at the gastrula stage. (D) Representative examples of control gastrulae, TSA-treated larvae, and rescued embryos at the same stage injected with the hd mRNA, respectively ordered from left to right. Note that in both the rescued embryos, the progeny of the blastomere that received hd was embedded into the ventral side. (E) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd-gfp mRNA, and the resulting embryos at the early blastula stage were disaggregated into individual cells that were eventually segregated into two populations, based on GFP fluorescence, by way of FACS. (F) Representative example of embryos expressing HD-GFP clonally, observed at the early blastula stage, just before dissociation. (G) Changes in gene expression level of nodal assessed by qPCR in whole embryos exposed to TSA, and in subpopulations of cells sorted from rescued TSA-treated embryos clonally expressing HD-GFP. Data are normalized and indicated as in Fig 1C.

Mentions: Following TSA treatment, phbox12-GFP expression started at a similar time with respect to control embryos. However, GFP fluorescence was detected in markedly larger and/or double patches throughout development of TSA-treated embryos (Fig 2D). Development of these embryos was rather normal until gastrulation (Fig 2D). By this time, control embryos displayed a clear DV polarity as shown by the thickening of the ventral side and the symmetric ventral-lateral arrangement of the two primary mesenchyme cell (PMC) clusters (Fig 2D). In striking contrast, more than 70% (n>500) of embryos exposed to TSA remained almost spherical, did not gastrulate, their mesenchyme cells were irregularly dispersed into the blastocoel, and skeletal elements were not mineralized (Figs 2D and 4D). Similar teratogenic effects on embryo development have been reported for other HDAC inhibitors [50–52].


Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo.

Cavalieri V, Spinelli G - PLoS ONE (2015)

Rescue of nodal transcription by HD expression into TSA-treated embryos.(A) 0.1–0.2 pg of the hd mRNA, or a control out-of-frame strim1 transcript [53], were injected into zygotes exposed to TSA and total RNA isolated from the resulting embryos at the early blastula stage. (B) qPCR measurements of relative nodal transcript abundance in embryos exposed to TSA and injected with increasing amounts of the hd mRNA, compared to the nodal mRNA level of control unperturbed embryos. Data are normalized and indicated as in Fig 1C. (C) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd mRNA together with the TRCD red fluorescent tracer, and the phenotype of the resulting embryos was examined at the gastrula stage. (D) Representative examples of control gastrulae, TSA-treated larvae, and rescued embryos at the same stage injected with the hd mRNA, respectively ordered from left to right. Note that in both the rescued embryos, the progeny of the blastomere that received hd was embedded into the ventral side. (E) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd-gfp mRNA, and the resulting embryos at the early blastula stage were disaggregated into individual cells that were eventually segregated into two populations, based on GFP fluorescence, by way of FACS. (F) Representative example of embryos expressing HD-GFP clonally, observed at the early blastula stage, just before dissociation. (G) Changes in gene expression level of nodal assessed by qPCR in whole embryos exposed to TSA, and in subpopulations of cells sorted from rescued TSA-treated embryos clonally expressing HD-GFP. Data are normalized and indicated as in Fig 1C.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664418&req=5

pone.0143860.g004: Rescue of nodal transcription by HD expression into TSA-treated embryos.(A) 0.1–0.2 pg of the hd mRNA, or a control out-of-frame strim1 transcript [53], were injected into zygotes exposed to TSA and total RNA isolated from the resulting embryos at the early blastula stage. (B) qPCR measurements of relative nodal transcript abundance in embryos exposed to TSA and injected with increasing amounts of the hd mRNA, compared to the nodal mRNA level of control unperturbed embryos. Data are normalized and indicated as in Fig 1C. (C) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd mRNA together with the TRCD red fluorescent tracer, and the phenotype of the resulting embryos was examined at the gastrula stage. (D) Representative examples of control gastrulae, TSA-treated larvae, and rescued embryos at the same stage injected with the hd mRNA, respectively ordered from left to right. Note that in both the rescued embryos, the progeny of the blastomere that received hd was embedded into the ventral side. (E) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd-gfp mRNA, and the resulting embryos at the early blastula stage were disaggregated into individual cells that were eventually segregated into two populations, based on GFP fluorescence, by way of FACS. (F) Representative example of embryos expressing HD-GFP clonally, observed at the early blastula stage, just before dissociation. (G) Changes in gene expression level of nodal assessed by qPCR in whole embryos exposed to TSA, and in subpopulations of cells sorted from rescued TSA-treated embryos clonally expressing HD-GFP. Data are normalized and indicated as in Fig 1C.
Mentions: Following TSA treatment, phbox12-GFP expression started at a similar time with respect to control embryos. However, GFP fluorescence was detected in markedly larger and/or double patches throughout development of TSA-treated embryos (Fig 2D). Development of these embryos was rather normal until gastrulation (Fig 2D). By this time, control embryos displayed a clear DV polarity as shown by the thickening of the ventral side and the symmetric ventral-lateral arrangement of the two primary mesenchyme cell (PMC) clusters (Fig 2D). In striking contrast, more than 70% (n>500) of embryos exposed to TSA remained almost spherical, did not gastrulate, their mesenchyme cells were irregularly dispersed into the blastocoel, and skeletal elements were not mineralized (Figs 2D and 4D). Similar teratogenic effects on embryo development have been reported for other HDAC inhibitors [50–52].

Bottom Line: Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae.Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae.Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Italy.

ABSTRACT
Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

Show MeSH
Related in: MedlinePlus