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A role for borg5 during trophectoderm differentiation.

Vong QP, Liu Z, Yoo JG, Chen R, Xie W, Sharov AA, Fan CM, Liu C, Ko MS, Zheng Y - Stem Cells (2010)

Bottom Line: In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction.It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts.Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland, USA.

ABSTRACT
Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.

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Expression of Borg5 after Oct3/4 downregulation. (A):Borg5 mRNA was upregulated in ZHBTc4 cells 12 hours after Tc addition as assayed by quantitative real-time polymerase chain reaction. (B):Borg5 mRNA level was upregulated after Oct3/4 siRNA treatment but not after Nanog or Sox2 siRNA treatments in E14 cells. The delayed Borg5 mRNA upregulation when compared with Tc addition in ZHBTc4 cells may reflect the slower and heterogeneous reduction of Oct3/4 mediated by siRNA. (C): Borg5 protein was upregulated 16 hours after Tc addition in both TE and TS culturing conditions based on Western blotting analysis. (D): Borg5 protein localization in ZHBTc4 ESCs and the differentiating ZHBTc4 cells 24 and 48 hours after Tc addition based on immunofluorescence staining. Oct3/4 and Cdx2 antibodies were used to label the undifferentiated and differentiating ESCs, respectively. Short and long arrows point to Borg5 in the cell process and along the edges of cells, respectively. (E): Borg5 protein is highly expressed in blastocyst-derived TSCs. (F): Borg5 protein localization in ESCs and TSCs. Merged images of Borg5 (red), Cdx2 (green), and DAPI (blue) are shown. Arrows point to Borg5 concentrated along the cell periphery of the TSC colony. Scale bars, 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; RNAi, RNA interference; siRNA, small interfering RNA; Tc, tetracycline; TE, trophectoderm; TS, trophoblast stem cell; TSC, trophoblast stem cell.
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fig02: Expression of Borg5 after Oct3/4 downregulation. (A):Borg5 mRNA was upregulated in ZHBTc4 cells 12 hours after Tc addition as assayed by quantitative real-time polymerase chain reaction. (B):Borg5 mRNA level was upregulated after Oct3/4 siRNA treatment but not after Nanog or Sox2 siRNA treatments in E14 cells. The delayed Borg5 mRNA upregulation when compared with Tc addition in ZHBTc4 cells may reflect the slower and heterogeneous reduction of Oct3/4 mediated by siRNA. (C): Borg5 protein was upregulated 16 hours after Tc addition in both TE and TS culturing conditions based on Western blotting analysis. (D): Borg5 protein localization in ZHBTc4 ESCs and the differentiating ZHBTc4 cells 24 and 48 hours after Tc addition based on immunofluorescence staining. Oct3/4 and Cdx2 antibodies were used to label the undifferentiated and differentiating ESCs, respectively. Short and long arrows point to Borg5 in the cell process and along the edges of cells, respectively. (E): Borg5 protein is highly expressed in blastocyst-derived TSCs. (F): Borg5 protein localization in ESCs and TSCs. Merged images of Borg5 (red), Cdx2 (green), and DAPI (blue) are shown. Arrows point to Borg5 concentrated along the cell periphery of the TSC colony. Scale bars, 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; RNAi, RNA interference; siRNA, small interfering RNA; Tc, tetracycline; TE, trophectoderm; TS, trophoblast stem cell; TSC, trophoblast stem cell.

Mentions: Using quantitative real-time polymerase chain reaction (qRT-PCR), we characterized the expression of Borg5 and found that Borg5 mRNA is strongly upregulated after reduction of Oct3/4 in both ZHBTc4 (Fig. 2A) and E14 ESCs (Fig. 2B), but not after reduction of Nanog or Sox2 (Fig. 2B). Therefore, Borg5 transcription is suppressed in ESCs and becomes upregulated specifically during TE differentiation. We analyzed previous whole genome ChIP-Seq studies of Oct3/4 binding sites in ESCs [18, 19] and found that there is no Oct3/4 binding site within 30 kb upstream and downstream of the transcriptional start site (TSS) of Borg5. Using ChIP-qPCR, we did not detect any Oct4 binding within 2 kb upstream of Borg5 TSS (data not shown). Thus, Oct3/4 is likely to indirectly suppress Borg5 expression in ESCs.


A role for borg5 during trophectoderm differentiation.

Vong QP, Liu Z, Yoo JG, Chen R, Xie W, Sharov AA, Fan CM, Liu C, Ko MS, Zheng Y - Stem Cells (2010)

Expression of Borg5 after Oct3/4 downregulation. (A):Borg5 mRNA was upregulated in ZHBTc4 cells 12 hours after Tc addition as assayed by quantitative real-time polymerase chain reaction. (B):Borg5 mRNA level was upregulated after Oct3/4 siRNA treatment but not after Nanog or Sox2 siRNA treatments in E14 cells. The delayed Borg5 mRNA upregulation when compared with Tc addition in ZHBTc4 cells may reflect the slower and heterogeneous reduction of Oct3/4 mediated by siRNA. (C): Borg5 protein was upregulated 16 hours after Tc addition in both TE and TS culturing conditions based on Western blotting analysis. (D): Borg5 protein localization in ZHBTc4 ESCs and the differentiating ZHBTc4 cells 24 and 48 hours after Tc addition based on immunofluorescence staining. Oct3/4 and Cdx2 antibodies were used to label the undifferentiated and differentiating ESCs, respectively. Short and long arrows point to Borg5 in the cell process and along the edges of cells, respectively. (E): Borg5 protein is highly expressed in blastocyst-derived TSCs. (F): Borg5 protein localization in ESCs and TSCs. Merged images of Borg5 (red), Cdx2 (green), and DAPI (blue) are shown. Arrows point to Borg5 concentrated along the cell periphery of the TSC colony. Scale bars, 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; RNAi, RNA interference; siRNA, small interfering RNA; Tc, tetracycline; TE, trophectoderm; TS, trophoblast stem cell; TSC, trophoblast stem cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2957878&req=5

fig02: Expression of Borg5 after Oct3/4 downregulation. (A):Borg5 mRNA was upregulated in ZHBTc4 cells 12 hours after Tc addition as assayed by quantitative real-time polymerase chain reaction. (B):Borg5 mRNA level was upregulated after Oct3/4 siRNA treatment but not after Nanog or Sox2 siRNA treatments in E14 cells. The delayed Borg5 mRNA upregulation when compared with Tc addition in ZHBTc4 cells may reflect the slower and heterogeneous reduction of Oct3/4 mediated by siRNA. (C): Borg5 protein was upregulated 16 hours after Tc addition in both TE and TS culturing conditions based on Western blotting analysis. (D): Borg5 protein localization in ZHBTc4 ESCs and the differentiating ZHBTc4 cells 24 and 48 hours after Tc addition based on immunofluorescence staining. Oct3/4 and Cdx2 antibodies were used to label the undifferentiated and differentiating ESCs, respectively. Short and long arrows point to Borg5 in the cell process and along the edges of cells, respectively. (E): Borg5 protein is highly expressed in blastocyst-derived TSCs. (F): Borg5 protein localization in ESCs and TSCs. Merged images of Borg5 (red), Cdx2 (green), and DAPI (blue) are shown. Arrows point to Borg5 concentrated along the cell periphery of the TSC colony. Scale bars, 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; RNAi, RNA interference; siRNA, small interfering RNA; Tc, tetracycline; TE, trophectoderm; TS, trophoblast stem cell; TSC, trophoblast stem cell.
Mentions: Using quantitative real-time polymerase chain reaction (qRT-PCR), we characterized the expression of Borg5 and found that Borg5 mRNA is strongly upregulated after reduction of Oct3/4 in both ZHBTc4 (Fig. 2A) and E14 ESCs (Fig. 2B), but not after reduction of Nanog or Sox2 (Fig. 2B). Therefore, Borg5 transcription is suppressed in ESCs and becomes upregulated specifically during TE differentiation. We analyzed previous whole genome ChIP-Seq studies of Oct3/4 binding sites in ESCs [18, 19] and found that there is no Oct3/4 binding site within 30 kb upstream and downstream of the transcriptional start site (TSS) of Borg5. Using ChIP-qPCR, we did not detect any Oct4 binding within 2 kb upstream of Borg5 TSS (data not shown). Thus, Oct3/4 is likely to indirectly suppress Borg5 expression in ESCs.

Bottom Line: In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction.It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts.Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland, USA.

ABSTRACT
Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.

Show MeSH
Related in: MedlinePlus