The Role of EphA Receptor Signaling in Neuroblast Survival During In Vitro Differentiation
Mouse embryonic stem cells can be differentiated into multiple cell types and can serve as an excellent model for studying developmental processes in vitro. In particular, stem cells can be differentiated into forebrain-like neurons, allowing investigation of nervous system development at single cell resolution. Eph receptor tyrosine kinases and their ephrin ligands play a critical role during in vivo cortical development, particularly during axon guidance. Preliminary data has shown that EphRs and ephrins are expressed during in vitro differentiation. In addition, we see EphA7 localization at the face of neural rosettes, where it co-expresses with markers of neuroblast identity. However, the role of Eph receptors and ephrins in neurogenesis is not well understood. Previous literature had shown that ephrin-A5 and EphA7 can function to balance cortical apoptosis during embryogenesis. We hypothesized that EphR/ephrin signaling may be required to balance apoptosis during in vitro neural development. A monolayer differentiation protocol was used to generate cells of forebrain fate at high yield. RNAi knockdown of EphA3, EphA4, and EphA7, was used to identify which Eph receptor was contributing to the apoptotic effect individually. It was found that transient knockdown of EphA3 and EphA4 fail to cause changes in apoptosis levels. However, knockdown of EphA7 at differentiation days 4 through 8 lead to a reduction in apoptosis suggesting that EphA7 can positively regulate cell death during differentiation. These data confirm that in vivo developmental events such as apoptosis can be modeled in an in vitro system. The innovative methods developed throughout the process of this research project may eventually prove to be useful in the analysis of other neurodevelopmental processes.
embryonic stem cell, in vitro differentiation, neurogenesis, ephrin, Eph receptor, apoptosis, siRNA, cell sorter, transfection, flow cytometry, Chemistry
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