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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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Differentiation of ZHBTc4 ESCs after Tc addition. (A): Addition of Tc induces rapid downregulation of Oct4 in both ZHBTc4 ESCs and ZHBTc4-H2B-green fluorescent protein ESCs. (B): Cdx2 protein appeared in the nuclei of differentiating ZHBTc4 ESCs 48 hours after Tc addition. At this time point many cells still expressed Nanog. Scale bar, 100 μm. (C): Tc addition induces distinct morphological changes in ZHBTc4 ESCs. Tc was added to the cells cultured in ES medium. Images were taken each day after Tc addition up to 8 days. Scale bar, 100 μm. (D, E): Reduction of Oct3/4 by Tc addition caused a significant increase in cell migration. Shown are distribution of distances migrated for >30 cells in each condition (D) and average distances migrated (E). Error bars, SEM. p value was calculated using Student's t test. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; Tc, tetracycline.
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fig01: Differentiation of ZHBTc4 ESCs after Tc addition. (A): Addition of Tc induces rapid downregulation of Oct4 in both ZHBTc4 ESCs and ZHBTc4-H2B-green fluorescent protein ESCs. (B): Cdx2 protein appeared in the nuclei of differentiating ZHBTc4 ESCs 48 hours after Tc addition. At this time point many cells still expressed Nanog. Scale bar, 100 μm. (C): Tc addition induces distinct morphological changes in ZHBTc4 ESCs. Tc was added to the cells cultured in ES medium. Images were taken each day after Tc addition up to 8 days. Scale bar, 100 μm. (D, E): Reduction of Oct3/4 by Tc addition caused a significant increase in cell migration. Shown are distribution of distances migrated for >30 cells in each condition (D) and average distances migrated (E). Error bars, SEM. p value was calculated using Student's t test. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; Tc, tetracycline.

Mentions: The above data suggest that specific regulators of cell morphogenesis might be upregulated very early to mediate unique cellular behaviors as ESCs differentiate into a specific lineage. Since cell motility is most pronounced during TE differentiation, we chose to focus on identifying TE specific morphological regulators responsible for TE cell motility using microarray analysis. We turned to the engineered ESCs, ZHBTc4, in which the pluripotency is maintained by a Tc-regulated Oct3/4 transgene [15], therefore TE differentiation can be induced more efficiently and homogeneously by Tc addition. Tc addition caused efficient reduction of Oct3/4 (Fig. 1A) and the appearance of lineage specific transcription factor Cdx2 (Fig. 1B). Consistent with RNAi of Oct3/4 in E14 ESCs, Tc addition caused enhanced migration of cell colonies toward each other followed by cell flattening (Fig. 1C–1E).


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)

Differentiation of ZHBTc4 ESCs after Tc addition. (A): Addition of Tc induces rapid downregulation of Oct4 in both ZHBTc4 ESCs and ZHBTc4-H2B-green fluorescent protein ESCs. (B): Cdx2 protein appeared in the nuclei of differentiating ZHBTc4 ESCs 48 hours after Tc addition. At this time point many cells still expressed Nanog. Scale bar, 100 μm. (C): Tc addition induces distinct morphological changes in ZHBTc4 ESCs. Tc was added to the cells cultured in ES medium. Images were taken each day after Tc addition up to 8 days. Scale bar, 100 μm. (D, E): Reduction of Oct3/4 by Tc addition caused a significant increase in cell migration. Shown are distribution of distances migrated for >30 cells in each condition (D) and average distances migrated (E). Error bars, SEM. p value was calculated using Student's t test. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; Tc, tetracycline.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Differentiation of ZHBTc4 ESCs after Tc addition. (A): Addition of Tc induces rapid downregulation of Oct4 in both ZHBTc4 ESCs and ZHBTc4-H2B-green fluorescent protein ESCs. (B): Cdx2 protein appeared in the nuclei of differentiating ZHBTc4 ESCs 48 hours after Tc addition. At this time point many cells still expressed Nanog. Scale bar, 100 μm. (C): Tc addition induces distinct morphological changes in ZHBTc4 ESCs. Tc was added to the cells cultured in ES medium. Images were taken each day after Tc addition up to 8 days. Scale bar, 100 μm. (D, E): Reduction of Oct3/4 by Tc addition caused a significant increase in cell migration. Shown are distribution of distances migrated for >30 cells in each condition (D) and average distances migrated (E). Error bars, SEM. p value was calculated using Student's t test. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; Tc, tetracycline.
Mentions: The above data suggest that specific regulators of cell morphogenesis might be upregulated very early to mediate unique cellular behaviors as ESCs differentiate into a specific lineage. Since cell motility is most pronounced during TE differentiation, we chose to focus on identifying TE specific morphological regulators responsible for TE cell motility using microarray analysis. We turned to the engineered ESCs, ZHBTc4, in which the pluripotency is maintained by a Tc-regulated Oct3/4 transgene [15], therefore TE differentiation can be induced more efficiently and homogeneously by Tc addition. Tc addition caused efficient reduction of Oct3/4 (Fig. 1A) and the appearance of lineage specific transcription factor Cdx2 (Fig. 1B). Consistent with RNAi of Oct3/4 in E14 ESCs, Tc addition caused enhanced migration of cell colonies toward each other followed by cell flattening (Fig. 1C–1E).

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