Increase in junctional G/F-actin ratio following Thymosin b4 depletion hinders collective cell migration during epidermal development

During mouse development, the epidermal sheet covers the eye through collective cell migration. Defects in this process, known as eyelid closure, may result in blindness. Collective migration requires coordination between polarity signals, cell adhesion, and the actin cytoskeleton. Here, we identified the G-actin binding protein, Thymosin b4 (encoded by Tmsb4x), as a major regulator of these processes. In utero depletion of Tmsb4x transcripts in mouse embryos did not affect epidermal proliferation, differentiation, or architecture; however, it hindered eyelid closure. We show that while individual keratinocytes can migrate without Thymosinb4 and apicobasal polarity is maintained, planar cell polarity (PCP) defects emerge without Thymosinb4 activity. Adherens junction dynamics are essential for PCP establishment, and while cell-cell adhesion is maintained without Thymosin b4, in vivo, and in vitro, adherens junction dynamic increases and stability decreases. We further show that while total actin levels are normal, junctional F-actin is downregulated, and G-actin is upregulated, leading to an increase in local G/F actin ratio and an increase in junctional actin dynamics. Our results provide novel insights into the spatial regulation of the actin pool and its importance during development.