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.

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.

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.

Borg5 regulates the motility of differentiating trophectoderm (TE) cells. (A):Borg5 small interfering RNA (siRNA) (S4 oligo) successfully suppressed Borg5 upregulation after addition of Tc. (B): Borg5 reduction by RNAi significantly inhibited cell migration induced by Tc. (C): Borg5 promotes actin protrusions in differentiating TE cells. Actin (red) and nuclei (green) were labeled with phalloidin and DAPI, 4′,6-diamidino-2-phenylindole respectively. Scale bar, 20 μm. The length of actin protrusion was measured from the centroid of each nucleus at the periphery of colonies to the tip of the longest actin protrusion from that cell at 48 hours after Tc addition. (D): Expressing Borg5 and Borg5-ins (insensitive to Borg5 S4 siRNA oligo) cDNA in ZHBTc4 cells in the presence or absence of Tc and S4 oligo as judged by Western blotting analysis. Borg5-ins, but not Borg5, cDNA could rescue Borg5 protein expression in the presence of S4 oligo. Borg5 protein was overexpressed to similar levels from Borg5 cDNA (in the absence of S4 oligo) or Borg5-ins cDNA (in the presence of S4 oligo) in the presence or absence of Tc. But this overexpression did not cause additional increase in cell motility (F). (E): Expression of Borg5-ins restored cell migration induced by Tc addition in the presence of S4 siRNA. (F): Elevated Borg5 expression in embryonic stem cells (ESCs) (−Tc) or in differentiating ESCs (+Tc) did not induce additional cell motility when compared with cells (vector controls) that did not have elevated Borg5. Therefore, overexpressing Borg5 did not cause a gain of function in cell motility. (G): Borg5 interacts with guanosine triphosphate (GTP)-bound Cdc42. FLAG-tagged Cdc42 wild-type (FLAG-Cdc42), GTP-bound Cdc42Q61L (FLAG-Q61L), or guanosine diphosphate-bound Cdc42T17N (FLAG-T17N) was expressed in the differentiating TE cells. Immunoprecipitations were carried out using Borg5 or FLAG antibodies. (H): Borg5 interacts with GTP-bound Cdc42 through its Cdc42/Rac interactive binding (CRIB) motif. FLAG-tagged forms of Cdc42 and Myc-tagged wild-type Borg5 (Myc-Borg5) or Myc-Borg5 with point mutations in the CRIB motif (Myc-Borg5-cribmut) were expressed. Immunoprecipitations using either immunoprecipitations using either Myc or FLAG antibodies or FLAG antibodies showed that the interaction between Cdc42Q61L and Borg5 is mediated by the CRIB motif of Borg5. (I): The CRIB motif of Borg5 is required for TE cell motility. Western-blotting analyses of Borg5 expression (wild-type Borg5 or Borg5-cribmut) in ZHBTc4 cells that were differentiating toward TE (+Tc) and treated with either control or Borg5 small interfering RNA. Expression of the RNAi-insensitive Borg5 containing mutations in the CRIB motif failed to rescue cell motility in the differentiating TE treated with Borg5 siRNA. The differently colored squares indicated the conditions for each treatment. Error bars, SEM. p values were calculated using Student's t test. Abbreviations: FLAG, DYKDDDDK octapeptide; RNAi, RNA interference; Tc, tetracycline.

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.