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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 promotes blastocyst formation. (A): Localization of Borg5 in the preimplantation embryos. Embryos from different stages were stained using antibodies to Borg5, aPKC, E-cadherin (E-cad), and Cdx2 as indicated. Nuclei were stained by DAPI. Borg5 localized to the cell–cell borders and cytoplasm. Quantifications revealed that many late morula and early blastocyst embryos had more Borg5 in outer cells than in inner cells (stronger outside). The number of embryos quantified in each group is shown on the histogram. (B): Quantitative real-time polymerase chain reaction analyses of Borg5 and Cdx2 expression in preimplantation embryos. Both Borg5 and Cdx2 are upregulated from eight-cell onward. M and B stands for morula and blastocyst, respectively. (C):Borg5 shRNA expression (S4-shRNA) reduced expression of Borg5 and Cdx2 mRNAs. Morula embryos were used. (D):Borg5 shRNA expression (S4-shRNA) reduced the formation of blastocysts when compared with control shRNA expression (con-shRNA). One, 2, 4, 8, M, and B in (B, D) refer to one, two, four, eight-cell, Morula, and blastocyst stage embryos, respectively. (E): Effects of Borg5 reduction on aPKC localization. aPKC typically exhibits both membrane (memb) and cytoplasmic localization in control shRNA-injected embryos. However, Borg5 (S4) shRNA injection resulted in embryos with largely cytoplasmic localization of aPKC (cyto). Quantification of embryos having normal membrane (memb), partial membrane (partial memb), or no membrane localization (cyto) is shown on the right. The number of embryos quantified in each group is shown on the histogram. Error bars, SEM. p value were calculated using Student's t test. Abbreviations: aPKC, atypical protein kinase C; shRNA, short-hairpin RNA.
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fig05: Borg5 promotes blastocyst formation. (A): Localization of Borg5 in the preimplantation embryos. Embryos from different stages were stained using antibodies to Borg5, aPKC, E-cadherin (E-cad), and Cdx2 as indicated. Nuclei were stained by DAPI. Borg5 localized to the cell–cell borders and cytoplasm. Quantifications revealed that many late morula and early blastocyst embryos had more Borg5 in outer cells than in inner cells (stronger outside). The number of embryos quantified in each group is shown on the histogram. (B): Quantitative real-time polymerase chain reaction analyses of Borg5 and Cdx2 expression in preimplantation embryos. Both Borg5 and Cdx2 are upregulated from eight-cell onward. M and B stands for morula and blastocyst, respectively. (C):Borg5 shRNA expression (S4-shRNA) reduced expression of Borg5 and Cdx2 mRNAs. Morula embryos were used. (D):Borg5 shRNA expression (S4-shRNA) reduced the formation of blastocysts when compared with control shRNA expression (con-shRNA). One, 2, 4, 8, M, and B in (B, D) refer to one, two, four, eight-cell, Morula, and blastocyst stage embryos, respectively. (E): Effects of Borg5 reduction on aPKC localization. aPKC typically exhibits both membrane (memb) and cytoplasmic localization in control shRNA-injected embryos. However, Borg5 (S4) shRNA injection resulted in embryos with largely cytoplasmic localization of aPKC (cyto). Quantification of embryos having normal membrane (memb), partial membrane (partial memb), or no membrane localization (cyto) is shown on the right. The number of embryos quantified in each group is shown on the histogram. Error bars, SEM. p value were calculated using Student's t test. Abbreviations: aPKC, atypical protein kinase C; shRNA, short-hairpin RNA.

Mentions: The above study led us to test whether Borg5 regulates blastocyst formation during preimplantation development. Immunofluorescence staining using affinity purified Borg5 antibodies generated in chicken revealed that Borg5 is localized along the cell–cell borders and in the cytoplasm in the post compaction embryos (Fig. 5A). Preabsorption using purified Borg5 protein showed that the staining was specific to the Borg5 protein (data not shown). From the late morula to blastocyst stage, many embryos had a higher amount of Borg5 in the outer cells than in the inner cells (Fig. 5A).


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 promotes blastocyst formation. (A): Localization of Borg5 in the preimplantation embryos. Embryos from different stages were stained using antibodies to Borg5, aPKC, E-cadherin (E-cad), and Cdx2 as indicated. Nuclei were stained by DAPI. Borg5 localized to the cell–cell borders and cytoplasm. Quantifications revealed that many late morula and early blastocyst embryos had more Borg5 in outer cells than in inner cells (stronger outside). The number of embryos quantified in each group is shown on the histogram. (B): Quantitative real-time polymerase chain reaction analyses of Borg5 and Cdx2 expression in preimplantation embryos. Both Borg5 and Cdx2 are upregulated from eight-cell onward. M and B stands for morula and blastocyst, respectively. (C):Borg5 shRNA expression (S4-shRNA) reduced expression of Borg5 and Cdx2 mRNAs. Morula embryos were used. (D):Borg5 shRNA expression (S4-shRNA) reduced the formation of blastocysts when compared with control shRNA expression (con-shRNA). One, 2, 4, 8, M, and B in (B, D) refer to one, two, four, eight-cell, Morula, and blastocyst stage embryos, respectively. (E): Effects of Borg5 reduction on aPKC localization. aPKC typically exhibits both membrane (memb) and cytoplasmic localization in control shRNA-injected embryos. However, Borg5 (S4) shRNA injection resulted in embryos with largely cytoplasmic localization of aPKC (cyto). Quantification of embryos having normal membrane (memb), partial membrane (partial memb), or no membrane localization (cyto) is shown on the right. The number of embryos quantified in each group is shown on the histogram. Error bars, SEM. p value were calculated using Student's t test. Abbreviations: aPKC, atypical protein kinase C; shRNA, short-hairpin RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Borg5 promotes blastocyst formation. (A): Localization of Borg5 in the preimplantation embryos. Embryos from different stages were stained using antibodies to Borg5, aPKC, E-cadherin (E-cad), and Cdx2 as indicated. Nuclei were stained by DAPI. Borg5 localized to the cell–cell borders and cytoplasm. Quantifications revealed that many late morula and early blastocyst embryos had more Borg5 in outer cells than in inner cells (stronger outside). The number of embryos quantified in each group is shown on the histogram. (B): Quantitative real-time polymerase chain reaction analyses of Borg5 and Cdx2 expression in preimplantation embryos. Both Borg5 and Cdx2 are upregulated from eight-cell onward. M and B stands for morula and blastocyst, respectively. (C):Borg5 shRNA expression (S4-shRNA) reduced expression of Borg5 and Cdx2 mRNAs. Morula embryos were used. (D):Borg5 shRNA expression (S4-shRNA) reduced the formation of blastocysts when compared with control shRNA expression (con-shRNA). One, 2, 4, 8, M, and B in (B, D) refer to one, two, four, eight-cell, Morula, and blastocyst stage embryos, respectively. (E): Effects of Borg5 reduction on aPKC localization. aPKC typically exhibits both membrane (memb) and cytoplasmic localization in control shRNA-injected embryos. However, Borg5 (S4) shRNA injection resulted in embryos with largely cytoplasmic localization of aPKC (cyto). Quantification of embryos having normal membrane (memb), partial membrane (partial memb), or no membrane localization (cyto) is shown on the right. The number of embryos quantified in each group is shown on the histogram. Error bars, SEM. p value were calculated using Student's t test. Abbreviations: aPKC, atypical protein kinase C; shRNA, short-hairpin RNA.
Mentions: The above study led us to test whether Borg5 regulates blastocyst formation during preimplantation development. Immunofluorescence staining using affinity purified Borg5 antibodies generated in chicken revealed that Borg5 is localized along the cell–cell borders and in the cytoplasm in the post compaction embryos (Fig. 5A). Preabsorption using purified Borg5 protein showed that the staining was specific to the Borg5 protein (data not shown). From the late morula to blastocyst stage, many embryos had a higher amount of Borg5 in the outer cells than in the inner cells (Fig. 5A).

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