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Enhancer of zeste acts as a major developmental regulator of Ciona intestinalis embryogenesis.

Le Goff E, Martinand-Mari C, Martin M, Feuillard J, Boublik Y, Godefroy N, Mangeat P, Baghdiguian S, Cavalli G - Biol Open (2015)

Bottom Line: These major phenotypic defects are specifically rescued by injection of a morpholino-resistant Ci-E(z) mRNA, which restores expression of Ci-E(z) protein and re-deposition of the H3K27me3 mark.As observed by qPCR analyses, Ci-E(z) invalidation leads to the early derepression of tissue-specific developmental genes, whereas late-acting developmental genes are generally down-regulated.Altogether, our results suggest that Ci-E(z) plays a major role during embryonic development in Ciona intestinalis by silencing early-acting developmental genes in a Hox-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Université Montpellier, Place Eugène Bataillon, Montpellier 34095, Cedex 5, France Institut des Sciences de l'Evolution (ISEM), UMR5554, CNRS, Montpellier 34095, France.

No MeSH data available.


Related in: MedlinePlus

Characterization of major phenotypic defects induced by Ci-E(z) invalidation at the 4-cell and hatching larva stages. (A,B) Ci-E(z) morphants are affected by cytokinesis defect at the 4-cell stage as demonstrated by TEM (A) and indirect immunofluorescence (B) analyses. The black arrowhead in A points to incomplete cell membrane formation. lv, lipid vesicle. (B) Actin (Phalloïdin, green) and DNA (DAPI, blue) double labeling. (C-F) Ultrastructural characterization of wild type (C) hatching larvae and Ci-E(z) morphants (D-F). (D) Semi-thin section of a Ci-E(z) morphant, stained with Toluidine Blue. The black rectangle window corresponds to the enlarged ultra-thin section TEM image shown in (F), showing abnormal accumulation of lipid vesicles (arrows) in muscle cells. Ultra-thin tail cross-sections of wild-type hatching larva (C) and Ci-E(z) morphant (E) showing the externally mislocalized muscle cell in Ci-E(z) morphants. e, epiderm cell; sm, striated muscle; ntc, notochord cell; nt, notochord lumina; n, nucleus. (G-J) Localization of neuronal form of tubulin in hatching larvae from control (G) and Ci-E(z) morphant (H). Acetylated-tubulin (red), actin (Phalloïdin, green) and DNA (DAPI, blue) triple labeling of Ciona intestinalis hatching larvae (G,H). Respective corresponding images (I,J) of acetylated-tubulin alone are shown in grey. The arrow indicates the position of nervous cells at the tail-head junction. Scale bars: 10 µm (C); 35 µm (D); 6 µm (F); 2 µm (A); 5 µm (B); 70 µm (H).
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BIO010835F3: Characterization of major phenotypic defects induced by Ci-E(z) invalidation at the 4-cell and hatching larva stages. (A,B) Ci-E(z) morphants are affected by cytokinesis defect at the 4-cell stage as demonstrated by TEM (A) and indirect immunofluorescence (B) analyses. The black arrowhead in A points to incomplete cell membrane formation. lv, lipid vesicle. (B) Actin (Phalloïdin, green) and DNA (DAPI, blue) double labeling. (C-F) Ultrastructural characterization of wild type (C) hatching larvae and Ci-E(z) morphants (D-F). (D) Semi-thin section of a Ci-E(z) morphant, stained with Toluidine Blue. The black rectangle window corresponds to the enlarged ultra-thin section TEM image shown in (F), showing abnormal accumulation of lipid vesicles (arrows) in muscle cells. Ultra-thin tail cross-sections of wild-type hatching larva (C) and Ci-E(z) morphant (E) showing the externally mislocalized muscle cell in Ci-E(z) morphants. e, epiderm cell; sm, striated muscle; ntc, notochord cell; nt, notochord lumina; n, nucleus. (G-J) Localization of neuronal form of tubulin in hatching larvae from control (G) and Ci-E(z) morphant (H). Acetylated-tubulin (red), actin (Phalloïdin, green) and DNA (DAPI, blue) triple labeling of Ciona intestinalis hatching larvae (G,H). Respective corresponding images (I,J) of acetylated-tubulin alone are shown in grey. The arrow indicates the position of nervous cells at the tail-head junction. Scale bars: 10 µm (C); 35 µm (D); 6 µm (F); 2 µm (A); 5 µm (B); 70 µm (H).

Mentions: The characterization of the Ci-E(z)-invalidated phenotype was further analyzed at 4-cell and hatching larval stages (Fig. 3). An extensive comparison between control and Ci-E(z) morphant embryos was performed. First, cells were characterized and identified at the ultrastructural level. In ultrathin sections, at the 4-cell stage, defects in cytokinesis with incomplete cell membrane formation (Fig. 3A), leading to multinucleated blastomeric cells (seen by immunofluorescence, Fig. 3B), were the striking earliest features of the Ci-E(z)-morphant phenotype. At the hatching larval stage, in contrast to control embryos (Fig. 3C), the relative positioning of cells was drastically affected in the morphant (Fig. 3E): the lack of notochord structure was particularly striking, pointing to the disorganization of embryonic tissues. In addition to the lack of notochord formation, a major feature of Ci-E(z) invalidation appeared to be a general defect in cell differentiation, which led to the generation of a majority of cells abnormally rich in lipid droplets, as evidenced by the toluidin blue staining in semi-thin sections of morphant cells (Fig. 3D). Mitochondria-enriched muscle cells were clearly identified as being externally delocalized compared with control tissue. These muscle cells were found located next to a set of undifferentiated cells containing numerous lipid droplets, presumably originating from endodermal precursor cells (Fig. 3E). Muscle cells appeared abnormally rich in lipid droplets as well (Fig. 3F). Last, a specific-antibody (acetylated-tubulin) was used to identify differentiated neural cells at the hatching larval stage. In control embryos, acetylated-tubulin stained cells helped specifying the localization of both the central (head-located part) and peripheral (regularly positioned all along the tail) nervous systems (Fig. 3G,I). In contrast, Ci-E(z) morphants displayed very few neural cells that were mislocalized at the head-tail junction (Fig. 3H,J). Moreover, the caudal nerve cord was totally lacking.Fig. 3.


Enhancer of zeste acts as a major developmental regulator of Ciona intestinalis embryogenesis.

Le Goff E, Martinand-Mari C, Martin M, Feuillard J, Boublik Y, Godefroy N, Mangeat P, Baghdiguian S, Cavalli G - Biol Open (2015)

Characterization of major phenotypic defects induced by Ci-E(z) invalidation at the 4-cell and hatching larva stages. (A,B) Ci-E(z) morphants are affected by cytokinesis defect at the 4-cell stage as demonstrated by TEM (A) and indirect immunofluorescence (B) analyses. The black arrowhead in A points to incomplete cell membrane formation. lv, lipid vesicle. (B) Actin (Phalloïdin, green) and DNA (DAPI, blue) double labeling. (C-F) Ultrastructural characterization of wild type (C) hatching larvae and Ci-E(z) morphants (D-F). (D) Semi-thin section of a Ci-E(z) morphant, stained with Toluidine Blue. The black rectangle window corresponds to the enlarged ultra-thin section TEM image shown in (F), showing abnormal accumulation of lipid vesicles (arrows) in muscle cells. Ultra-thin tail cross-sections of wild-type hatching larva (C) and Ci-E(z) morphant (E) showing the externally mislocalized muscle cell in Ci-E(z) morphants. e, epiderm cell; sm, striated muscle; ntc, notochord cell; nt, notochord lumina; n, nucleus. (G-J) Localization of neuronal form of tubulin in hatching larvae from control (G) and Ci-E(z) morphant (H). Acetylated-tubulin (red), actin (Phalloïdin, green) and DNA (DAPI, blue) triple labeling of Ciona intestinalis hatching larvae (G,H). Respective corresponding images (I,J) of acetylated-tubulin alone are shown in grey. The arrow indicates the position of nervous cells at the tail-head junction. Scale bars: 10 µm (C); 35 µm (D); 6 µm (F); 2 µm (A); 5 µm (B); 70 µm (H).
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BIO010835F3: Characterization of major phenotypic defects induced by Ci-E(z) invalidation at the 4-cell and hatching larva stages. (A,B) Ci-E(z) morphants are affected by cytokinesis defect at the 4-cell stage as demonstrated by TEM (A) and indirect immunofluorescence (B) analyses. The black arrowhead in A points to incomplete cell membrane formation. lv, lipid vesicle. (B) Actin (Phalloïdin, green) and DNA (DAPI, blue) double labeling. (C-F) Ultrastructural characterization of wild type (C) hatching larvae and Ci-E(z) morphants (D-F). (D) Semi-thin section of a Ci-E(z) morphant, stained with Toluidine Blue. The black rectangle window corresponds to the enlarged ultra-thin section TEM image shown in (F), showing abnormal accumulation of lipid vesicles (arrows) in muscle cells. Ultra-thin tail cross-sections of wild-type hatching larva (C) and Ci-E(z) morphant (E) showing the externally mislocalized muscle cell in Ci-E(z) morphants. e, epiderm cell; sm, striated muscle; ntc, notochord cell; nt, notochord lumina; n, nucleus. (G-J) Localization of neuronal form of tubulin in hatching larvae from control (G) and Ci-E(z) morphant (H). Acetylated-tubulin (red), actin (Phalloïdin, green) and DNA (DAPI, blue) triple labeling of Ciona intestinalis hatching larvae (G,H). Respective corresponding images (I,J) of acetylated-tubulin alone are shown in grey. The arrow indicates the position of nervous cells at the tail-head junction. Scale bars: 10 µm (C); 35 µm (D); 6 µm (F); 2 µm (A); 5 µm (B); 70 µm (H).
Mentions: The characterization of the Ci-E(z)-invalidated phenotype was further analyzed at 4-cell and hatching larval stages (Fig. 3). An extensive comparison between control and Ci-E(z) morphant embryos was performed. First, cells were characterized and identified at the ultrastructural level. In ultrathin sections, at the 4-cell stage, defects in cytokinesis with incomplete cell membrane formation (Fig. 3A), leading to multinucleated blastomeric cells (seen by immunofluorescence, Fig. 3B), were the striking earliest features of the Ci-E(z)-morphant phenotype. At the hatching larval stage, in contrast to control embryos (Fig. 3C), the relative positioning of cells was drastically affected in the morphant (Fig. 3E): the lack of notochord structure was particularly striking, pointing to the disorganization of embryonic tissues. In addition to the lack of notochord formation, a major feature of Ci-E(z) invalidation appeared to be a general defect in cell differentiation, which led to the generation of a majority of cells abnormally rich in lipid droplets, as evidenced by the toluidin blue staining in semi-thin sections of morphant cells (Fig. 3D). Mitochondria-enriched muscle cells were clearly identified as being externally delocalized compared with control tissue. These muscle cells were found located next to a set of undifferentiated cells containing numerous lipid droplets, presumably originating from endodermal precursor cells (Fig. 3E). Muscle cells appeared abnormally rich in lipid droplets as well (Fig. 3F). Last, a specific-antibody (acetylated-tubulin) was used to identify differentiated neural cells at the hatching larval stage. In control embryos, acetylated-tubulin stained cells helped specifying the localization of both the central (head-located part) and peripheral (regularly positioned all along the tail) nervous systems (Fig. 3G,I). In contrast, Ci-E(z) morphants displayed very few neural cells that were mislocalized at the head-tail junction (Fig. 3H,J). Moreover, the caudal nerve cord was totally lacking.Fig. 3.

Bottom Line: These major phenotypic defects are specifically rescued by injection of a morpholino-resistant Ci-E(z) mRNA, which restores expression of Ci-E(z) protein and re-deposition of the H3K27me3 mark.As observed by qPCR analyses, Ci-E(z) invalidation leads to the early derepression of tissue-specific developmental genes, whereas late-acting developmental genes are generally down-regulated.Altogether, our results suggest that Ci-E(z) plays a major role during embryonic development in Ciona intestinalis by silencing early-acting developmental genes in a Hox-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Université Montpellier, Place Eugène Bataillon, Montpellier 34095, Cedex 5, France Institut des Sciences de l'Evolution (ISEM), UMR5554, CNRS, Montpellier 34095, France.

No MeSH data available.


Related in: MedlinePlus