title: Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation.
abstract: Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a 'developmental clock' using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model.
authors.affiliation: Department of Biochemistry and Molecular Biology, Tel Aviv University, Israel.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Boxman
authors.affiliation: Department of Biochemistry and Molecular Biology, Tel Aviv University, Israel.
authors.forename: Naor
authors.initials: N
authors.lastname: Sagy
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
authors.forename: Sirisha
authors.initials: S
authors.lastname: Achanta
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
authors.forename: Rajanikanth
authors.initials: R
authors.lastname: Vadigepalli
authors.affiliation: Department of Biochemistry and Molecular Biology, Tel Aviv University, Israel.
authors.forename: Iftach
authors.initials: I
authors.lastname: Nachman
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D058732:Embryoid Bodies / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D005326:Fetal Proteins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000235:genetics; D000098908:Brachyury Protein
chemical_list: D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein