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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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Borg5 regulates Cdx2 expression and trophectoderm (TE) cell sorting. (A): ZHBTc4-Con and ZHBTc4-S4 cells both downregulate Oct3/4 after Tc addition, but only ZHBTc4-Con cells upregulated Borg5 after Tc addition as judged by Western blotting. (B): Reduction of Borg5 by S4-shRNA caused reduction of Cdx2 mRNA expression in ZHBTc4 cells after Tc addition. (C): Reduction of Cdx2 expression by two different small interfering RNA oligos resulted in a reduction of Borg5 mRNA expression in ZHBTc4 cells after Tc addition. (D): Borg5 interacts with aPKC in differentiating TE cells. Antibodies (Ab) to Borg5, aPKC, or control IgG (MockAb) were used for reciprocal immunoprecipitations in lysates made from the differentiating TE cells. (E): Images of E14 ESCs mixed with either ZHBTc4-Con or ZHBTc4-S4 ESCs expressing GFP (green). Oct3/4 antibodies (red) stained pluripotent ESCs. (F): Examples of colonies with TE cells repositioned to the outside of the colonies 42–48 hours after Tc addition. The differentiating ZHBTc4 cells are labeled green with GFP whereas the pluripotent E14 ESCs are labeled red by Oct3/4. (G): Examples of a scattered cell colony in which the differentiating green ZHBTc4 cells are scattered throughout the E14 ESCs (red) and an inside cell colony with the differentiating green ZHBTc4 cells enclosed inside the E14 ESCs (red). Scale bars in (E–G), 100 μm. (H): Quantification of colonies that have TE cells outside, scattered, or inside. Error bars, SEM. p values were calculated by Student's t test. Abbreviations: aPKC, atypical protein kinase C; RNAi, RNA interference; shRNA, short-hairpin RNA; Tc, tetracycline.
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fig04: Borg5 regulates Cdx2 expression and trophectoderm (TE) cell sorting. (A): ZHBTc4-Con and ZHBTc4-S4 cells both downregulate Oct3/4 after Tc addition, but only ZHBTc4-Con cells upregulated Borg5 after Tc addition as judged by Western blotting. (B): Reduction of Borg5 by S4-shRNA caused reduction of Cdx2 mRNA expression in ZHBTc4 cells after Tc addition. (C): Reduction of Cdx2 expression by two different small interfering RNA oligos resulted in a reduction of Borg5 mRNA expression in ZHBTc4 cells after Tc addition. (D): Borg5 interacts with aPKC in differentiating TE cells. Antibodies (Ab) to Borg5, aPKC, or control IgG (MockAb) were used for reciprocal immunoprecipitations in lysates made from the differentiating TE cells. (E): Images of E14 ESCs mixed with either ZHBTc4-Con or ZHBTc4-S4 ESCs expressing GFP (green). Oct3/4 antibodies (red) stained pluripotent ESCs. (F): Examples of colonies with TE cells repositioned to the outside of the colonies 42–48 hours after Tc addition. The differentiating ZHBTc4 cells are labeled green with GFP whereas the pluripotent E14 ESCs are labeled red by Oct3/4. (G): Examples of a scattered cell colony in which the differentiating green ZHBTc4 cells are scattered throughout the E14 ESCs (red) and an inside cell colony with the differentiating green ZHBTc4 cells enclosed inside the E14 ESCs (red). Scale bars in (E–G), 100 μm. (H): Quantification of colonies that have TE cells outside, scattered, or inside. Error bars, SEM. p values were calculated by Student's t test. Abbreviations: aPKC, atypical protein kinase C; RNAi, RNA interference; shRNA, short-hairpin RNA; Tc, tetracycline.

Mentions: Since Borg5 is upregulated during TE differentiation as early as Cdx2, we asked whether it could influence Cdx2 expression. We created ZHBTc4 ESC lines that stably express either S4 short-hairpin RNA (shRNA) or control shRNA (Supporting Information). Both cell lines expressed Oct3/4 and were responsive to Tc, but ZHBTc4-S4 cells failed to upregulate Borg5 as expected (Fig. 4A). qRT-PCR analyses showed that inhibiting Borg5 during TE differentiation caused a corresponding reduction of Cdx2 mRNA expression in these cells (Fig. 4B). Interestingly, reduction of Cdx2 expression using two siRNA oligos targeting different regions of Cdx2 also caused a corresponding reduction of Borg5 mRNA expression (Fig. 4C).


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)

Borg5 regulates Cdx2 expression and trophectoderm (TE) cell sorting. (A): ZHBTc4-Con and ZHBTc4-S4 cells both downregulate Oct3/4 after Tc addition, but only ZHBTc4-Con cells upregulated Borg5 after Tc addition as judged by Western blotting. (B): Reduction of Borg5 by S4-shRNA caused reduction of Cdx2 mRNA expression in ZHBTc4 cells after Tc addition. (C): Reduction of Cdx2 expression by two different small interfering RNA oligos resulted in a reduction of Borg5 mRNA expression in ZHBTc4 cells after Tc addition. (D): Borg5 interacts with aPKC in differentiating TE cells. Antibodies (Ab) to Borg5, aPKC, or control IgG (MockAb) were used for reciprocal immunoprecipitations in lysates made from the differentiating TE cells. (E): Images of E14 ESCs mixed with either ZHBTc4-Con or ZHBTc4-S4 ESCs expressing GFP (green). Oct3/4 antibodies (red) stained pluripotent ESCs. (F): Examples of colonies with TE cells repositioned to the outside of the colonies 42–48 hours after Tc addition. The differentiating ZHBTc4 cells are labeled green with GFP whereas the pluripotent E14 ESCs are labeled red by Oct3/4. (G): Examples of a scattered cell colony in which the differentiating green ZHBTc4 cells are scattered throughout the E14 ESCs (red) and an inside cell colony with the differentiating green ZHBTc4 cells enclosed inside the E14 ESCs (red). Scale bars in (E–G), 100 μm. (H): Quantification of colonies that have TE cells outside, scattered, or inside. Error bars, SEM. p values were calculated by Student's t test. Abbreviations: aPKC, atypical protein kinase C; RNAi, RNA interference; shRNA, short-hairpin RNA; Tc, tetracycline.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Borg5 regulates Cdx2 expression and trophectoderm (TE) cell sorting. (A): ZHBTc4-Con and ZHBTc4-S4 cells both downregulate Oct3/4 after Tc addition, but only ZHBTc4-Con cells upregulated Borg5 after Tc addition as judged by Western blotting. (B): Reduction of Borg5 by S4-shRNA caused reduction of Cdx2 mRNA expression in ZHBTc4 cells after Tc addition. (C): Reduction of Cdx2 expression by two different small interfering RNA oligos resulted in a reduction of Borg5 mRNA expression in ZHBTc4 cells after Tc addition. (D): Borg5 interacts with aPKC in differentiating TE cells. Antibodies (Ab) to Borg5, aPKC, or control IgG (MockAb) were used for reciprocal immunoprecipitations in lysates made from the differentiating TE cells. (E): Images of E14 ESCs mixed with either ZHBTc4-Con or ZHBTc4-S4 ESCs expressing GFP (green). Oct3/4 antibodies (red) stained pluripotent ESCs. (F): Examples of colonies with TE cells repositioned to the outside of the colonies 42–48 hours after Tc addition. The differentiating ZHBTc4 cells are labeled green with GFP whereas the pluripotent E14 ESCs are labeled red by Oct3/4. (G): Examples of a scattered cell colony in which the differentiating green ZHBTc4 cells are scattered throughout the E14 ESCs (red) and an inside cell colony with the differentiating green ZHBTc4 cells enclosed inside the E14 ESCs (red). Scale bars in (E–G), 100 μm. (H): Quantification of colonies that have TE cells outside, scattered, or inside. Error bars, SEM. p values were calculated by Student's t test. Abbreviations: aPKC, atypical protein kinase C; RNAi, RNA interference; shRNA, short-hairpin RNA; Tc, tetracycline.
Mentions: Since Borg5 is upregulated during TE differentiation as early as Cdx2, we asked whether it could influence Cdx2 expression. We created ZHBTc4 ESC lines that stably express either S4 short-hairpin RNA (shRNA) or control shRNA (Supporting Information). Both cell lines expressed Oct3/4 and were responsive to Tc, but ZHBTc4-S4 cells failed to upregulate Borg5 as expected (Fig. 4A). qRT-PCR analyses showed that inhibiting Borg5 during TE differentiation caused a corresponding reduction of Cdx2 mRNA expression in these cells (Fig. 4B). Interestingly, reduction of Cdx2 expression using two siRNA oligos targeting different regions of Cdx2 also caused a corresponding reduction of Borg5 mRNA expression (Fig. 4C).

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