Emergence of an Apical Epithelial Cell Surface In Vivo

Abstract

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological and laser-dissection experiments with theoretical modelling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

“…2C,D; Table S1, Movie 1). These pulses were similar to those observed after disruption of Fmn1 function; importantly, however, expression of DN-RhoA rarely elicited the complete collapse of the apical cell surface that is associated with formin loss in these cells (Table S1) (Sedzinski et al, 2016). This difference might result from the efficacy of the reagents [DN-RhoA versus a morpholino against Fmn1 (Fmn1-MO)] in decreasing the actin-based pushing forces, or it could relate to the specific functions of the two proteins in controlling force balance at the MCC apical surface, as we discuss below.…”

“…Normally, the apical surfaces of MCCs are rounded (low kurtosis) during early emergence, indicating that pushing forces dominate at these stages (Figs 2A and 3B, black) (Sedzinski et al, 2016). By contrast, apical domain shapes of MCCs expressing DNRhoA were more polygonal, with elongated cell-cell boundaries meeting at sharp junctional angles (Figs 2C,3B,pink).…”

“…RhoA controls the rate of MCC apical actin network assembly MCC emergence is characterized by a strong positive correlation between apical area and apical actin concentration, suggesting that the apical actin network assembly drives emergence (Sedzinski et al, 2016). We therefore assessed the effect of RhoA manipulation on actin network assembly in MCCs by measuring apical actin concentration over time ( Fig.…”

“…Given the requirement for Fmn1 in apical emergence (Sedzinski et al, 2016), we probed the role of RhoA in this process. We first measured the dynamics of RhoA activity using the active RhoA biosensor (rGBD), which has been shown previously to be effective in Xenopus (for examples, see Benink and Bement, 2005;Breznau et al, 2015;Reyes et al, 2014).…”

“…An outline of the molecular framework for the control of radial intercalation of MCCs is now emerging, revealing key roles for dystroglycan, Rab11, the Par complex, Slit2 and the Rfx2 transcription factor (Chung et al, 2014;Kim et al, 2012;Sirour et al, 2011;Werner et al, 2014). In addition, we have recently explored the mechanical basis specifically of apical surface emergence in nascent MCCs, finding that the forces that drive apical emergence are cellautonomous and dependent on the assembly of an apical actin network generating effective two-dimensional (2D) pushing forces (Sedzinski et al, 2016).…”