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Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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Developmental defects induced by pancIl17d knockdown and rescue by addition of recombinant IL17D protein. (A) TUNEL assay of morula embryos. Arrowheads indicate TUNEL-positive blastomeres. pancIl17d knockdown embryos showed increased TUNEL-positive cells. Two representative blastomeres are shown. To the right is a box plot of the number of TUNEL-positive cells in each embryo. (B) Morphology of late blastocysts. (C) Expression levels of pancIl17d and Il17d measured by qPCR in control, pancIl17d knockdown and rIL17D-supplemented pancIl17d knockdown morula embryos. (D) Survival rate of control and pancIl17d knockdown embryos at day 4.5 of in vitro culture. Two different siRNAs targeting pancIl17d were used. (E) Scatter plots of gene expression in control and pancIl17d knockdown morula embryos based on the RPKMs of RefSeq genes. Red dots indicate the genes that show statistically significant changes. (F) Immunostaining of CDX2 protein in control and pancIl17d knockdown late blastocyst. Arrowheads indicate CDX2-negative outer cells Beneath is a box plot of the number of CDX2-negative outer cells in each embryo *P<0.05; ***P<0.001. Error bars indicate s.e.m.
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DEV116996F4: Developmental defects induced by pancIl17d knockdown and rescue by addition of recombinant IL17D protein. (A) TUNEL assay of morula embryos. Arrowheads indicate TUNEL-positive blastomeres. pancIl17d knockdown embryos showed increased TUNEL-positive cells. Two representative blastomeres are shown. To the right is a box plot of the number of TUNEL-positive cells in each embryo. (B) Morphology of late blastocysts. (C) Expression levels of pancIl17d and Il17d measured by qPCR in control, pancIl17d knockdown and rIL17D-supplemented pancIl17d knockdown morula embryos. (D) Survival rate of control and pancIl17d knockdown embryos at day 4.5 of in vitro culture. Two different siRNAs targeting pancIl17d were used. (E) Scatter plots of gene expression in control and pancIl17d knockdown morula embryos based on the RPKMs of RefSeq genes. Red dots indicate the genes that show statistically significant changes. (F) Immunostaining of CDX2 protein in control and pancIl17d knockdown late blastocyst. Arrowheads indicate CDX2-negative outer cells Beneath is a box plot of the number of CDX2-negative outer cells in each embryo *P<0.05; ***P<0.001. Error bars indicate s.e.m.

Mentions: Since the effect of pancIl17d knockdown was drastic, we focused on investigating the roles of this pancRNA in embryonic development. Many pancIl17d knockdown embryos died between the 8-cell and early blastocyst stages. To establish whether cell death was enhanced in the pancIl17d knockdown embryos, we performed TdT-mediated dUTP nick-end labeling (TUNEL) staining of the pancIl17d knockdown embryos at the morula stage (Fig. 4A). Consistent with a previous report (Brison and Schultz, 1997), control embryos underwent little apoptosis during blastocyst formation. By contrast, pancIl17d knockdown embryos exhibited multiple TUNEL-positive blastomeres, suggesting that many pancIl17d knockdown embryos died by the blastocyst stage due to excessive apoptosis. It is noteworthy that the developmental capacity to form a blastocyst was restored when recombinant mouse IL17D protein (rIL17D) was added to the medium at the 4-cell stage, although the knockdown effect continued until the morula stage (Fig. 4B,C). The addition of rIL17D significantly increased the rate of success of blastocyst formation in pancIl17d knockdown embryos (from 21.5±2.7% to 62.1±5.9%; Fig. 4D). These results suggest that pancIl17d plays an important role in blastocyst formation by upregulating the partner gene.Fig. 4.


Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Developmental defects induced by pancIl17d knockdown and rescue by addition of recombinant IL17D protein. (A) TUNEL assay of morula embryos. Arrowheads indicate TUNEL-positive blastomeres. pancIl17d knockdown embryos showed increased TUNEL-positive cells. Two representative blastomeres are shown. To the right is a box plot of the number of TUNEL-positive cells in each embryo. (B) Morphology of late blastocysts. (C) Expression levels of pancIl17d and Il17d measured by qPCR in control, pancIl17d knockdown and rIL17D-supplemented pancIl17d knockdown morula embryos. (D) Survival rate of control and pancIl17d knockdown embryos at day 4.5 of in vitro culture. Two different siRNAs targeting pancIl17d were used. (E) Scatter plots of gene expression in control and pancIl17d knockdown morula embryos based on the RPKMs of RefSeq genes. Red dots indicate the genes that show statistically significant changes. (F) Immunostaining of CDX2 protein in control and pancIl17d knockdown late blastocyst. Arrowheads indicate CDX2-negative outer cells Beneath is a box plot of the number of CDX2-negative outer cells in each embryo *P<0.05; ***P<0.001. Error bars indicate s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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DEV116996F4: Developmental defects induced by pancIl17d knockdown and rescue by addition of recombinant IL17D protein. (A) TUNEL assay of morula embryos. Arrowheads indicate TUNEL-positive blastomeres. pancIl17d knockdown embryos showed increased TUNEL-positive cells. Two representative blastomeres are shown. To the right is a box plot of the number of TUNEL-positive cells in each embryo. (B) Morphology of late blastocysts. (C) Expression levels of pancIl17d and Il17d measured by qPCR in control, pancIl17d knockdown and rIL17D-supplemented pancIl17d knockdown morula embryos. (D) Survival rate of control and pancIl17d knockdown embryos at day 4.5 of in vitro culture. Two different siRNAs targeting pancIl17d were used. (E) Scatter plots of gene expression in control and pancIl17d knockdown morula embryos based on the RPKMs of RefSeq genes. Red dots indicate the genes that show statistically significant changes. (F) Immunostaining of CDX2 protein in control and pancIl17d knockdown late blastocyst. Arrowheads indicate CDX2-negative outer cells Beneath is a box plot of the number of CDX2-negative outer cells in each embryo *P<0.05; ***P<0.001. Error bars indicate s.e.m.
Mentions: Since the effect of pancIl17d knockdown was drastic, we focused on investigating the roles of this pancRNA in embryonic development. Many pancIl17d knockdown embryos died between the 8-cell and early blastocyst stages. To establish whether cell death was enhanced in the pancIl17d knockdown embryos, we performed TdT-mediated dUTP nick-end labeling (TUNEL) staining of the pancIl17d knockdown embryos at the morula stage (Fig. 4A). Consistent with a previous report (Brison and Schultz, 1997), control embryos underwent little apoptosis during blastocyst formation. By contrast, pancIl17d knockdown embryos exhibited multiple TUNEL-positive blastomeres, suggesting that many pancIl17d knockdown embryos died by the blastocyst stage due to excessive apoptosis. It is noteworthy that the developmental capacity to form a blastocyst was restored when recombinant mouse IL17D protein (rIL17D) was added to the medium at the 4-cell stage, although the knockdown effect continued until the morula stage (Fig. 4B,C). The addition of rIL17D significantly increased the rate of success of blastocyst formation in pancIl17d knockdown embryos (from 21.5±2.7% to 62.1±5.9%; Fig. 4D). These results suggest that pancIl17d plays an important role in blastocyst formation by upregulating the partner gene.Fig. 4.

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Show MeSH
Related in: MedlinePlus