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A role for borg5 during trophectoderm differentiation.

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.

SUBMITTER: Vong QP

PROVIDER: S-EPMC2957878 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:STUDY QUESTION:What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER:AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY:AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION:Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 ?M) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 ?M AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS:Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE:Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA:Not applicable. LIMITATIONS, REASONS FOR CAUTION:Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS:Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S):Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.

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A role for borg5 during trophectoderm differentiation.

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