Abstract
The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses.
Highlights
The three-dimensional (3D) morphologies of ectodermal organs are required for their inherent physiological and physical functions and are formed by an accumulation of highly dynamic cell behaviors during embryonic development [1,2,3]
We found that phosphorylated cofilin (p-cofilin), which is a member of the actin-depolymerizing factor (ADF)/cofilin family, was localized in the growth-arrested primary enamel knot (EK) and dental lamina (DL) regions of the tooth germ (Fig 4A, S7A Fig)
We found that LIM-kinase 2 (Limk2) but not LIM-Kinase 1 (Limk1) or Testis-Specific Kinase 1 (Tesk1, a member of the LIM kinase family that phosphorylates members of the ADF/cofilin family) was expressed at relatively high levels in the DL and EK in which growth was arrested
Summary
The three-dimensional (3D) morphologies of ectodermal organs are required for their inherent physiological and physical functions and are formed by an accumulation of highly dynamic cell behaviors during embryonic development [1,2,3]. Spatiotemporal regulation and the combination of epithelial deformation modes, including invagination, lumen formation, branching, and anisotropic tissue expansion/elongation, determine the final organ shape [1, 2, 4, 5]. The molecular genetic approach has revealed dozens of genes that are involved in the regulation of epithelial tissue formation. The actin cytoskeleton that is coordinated by actin-depolymerizing factor (ADF)/cofilin has been implicated in cell shape, motility and proliferation in response to external stimuli and intracellular signals [6,7,8]. How the spatiotemporal changes in cellular behavior and signaling events cooperate to regulate local deformations and contribute to the overall 3D organ shape during dynamic morphogenetic processes remains unknown. To understand the regulatory mechanisms underlying organ morphogenesis, it is essential to visualize and track the cellular dynamics as well as the signaling events at a single-cell resolution [11]
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