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Organized cell movement is a major mechanism underlying facial morphogenesis

University of British Columbia

The mechanisms of embryonic facial morphogenesis are poorly understood because direct visualization of the growing embryo is challenging. We refined an organ culture method to visualize the individual cells within the frontonasal mass at high resolution. The fate of the frontonasal mass is to form the premaxilla, nasal septum and facial midline. During normal development the frontonasal mass begins as wide prominence but then the nasal pits relocate to the midline, causing dramatic medio-lateral narrowing. We confirmed that in vitro, the frontonasal mass narrowed over a 48h period, similar to in vivo. Removing the eyes, brain and most of the surrounding face did not impede facial narrowing suggesting intrinsic rather than extrinsic mechanisms were involved. Indeed, blocking the cytoskeletal rearrangements with the Rho GTPase inhibitor, Y27632 (ROCKi) completely inhibited narrowing. Organ cultures were stained with Hoechst dye and imaged using confocal microscopy. Nuclei were imaged for 4-6 hours at 10 minute intervals. Manual cell tracking was carried out across the frontonasal mass. At 10X magnification, striking patterns of order, disorder and then order in vectors of movement were observed. In ROCKi treated cultures there was disorder for the entire culture period. Clustering the vectors according to similarity of the angle revealed large groups of cells were moving in a similar manner in the controls but in the ROCKi treated cultures, clusters were poorly defined and smaller in size. In order to assess symmetry and patterns of divergence and convergence the vector data was interpolated over an evenly spaced grid. The controls had a high degree of right-left symmetry whereas the ROCKi-treated cultures lacked symmetry. We also examined the data to look for sources or sinks in the cell movements. Divergence and convergence bands were located in the mediolateral axis with branches at the lateral edges. The change in direction from convergence to divergence and then back to convergence was rapid, often switching within 20 minutes. This rapid cycling is on the same scale as GTPase switching. Our data suggest that orchestrated mesenchymal cell behaviors, mediated by Rho GTPases are involved in convergent-extension in the face.

Text

2019-09-04

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/71599

Craniofacial Science

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/