Title: Directed Mesenchymal Cell Movements Contribute to Midfacial Narrowing
Adrian Danescu (Presenter)
University of British Columbia
Jaspreet Rekhi, University of British Columbia
Joy Richman, University of British Columbia
Objectives: We investigated a critical time during craniofacial development, the narrowing of the midface, in a chicken embryo model. Multiple syndromes and non-syndromic conditions include abnormal narrowing of the face with hypertelorism, broad nasal bridge and premaxillary hypoplasia. We examined the extrinsic versus intrinsic factors contributing to midline narrowing
Methods: The frontonasal mass and lateral nasal prominences were dissected as one piece from stage 25 embryos. A subset of cultures were treated with ROCK inhibitor (10 µM Y27632). Organ cultures were photographed every 24h for 48h and the width between the nasal slits was measured. Other cultures were used for time-lapse confocal microscopy using Hoechst dye to visualize the nuclei. Movies were captured at10 minute intervals for 6h.
Results: Narrowing was reproduced in culture independent of whether eyes and brain were included (n = 13). Y27632 completely blocked narrowing (n = 12). Time-lapse cell tracking of labeled nuclei at 10X (n=3) showed the whole frontonasal mass contracted in the first 3 hours and then expanded in a cranial caudal direction. At 30X (n=3) the lateral third of the frontonasal mass was captured. Nuclei moved cranially and caudally, away from the midline. ROCKi reduced the magnitude and directionality of the cell movements. At 63X (n=3) it was clear that cells next to the nasal slit were moving medially whereas the nuclei that were more medial were moving towards the nasal slit, in other words converging. Y27632 caused most cells to remain stationary for 6h.
Conclusions: Y27632 blocked all intrinsic movement by interfering with the cytoskeleton. Since treatment of the frontonasal mass blocks narrowing, we conclude that intrinsic cellular migration is the main mechanism underlying rapid tissue morphogenesis in the midface. Our novel live imaging methods allow direct observation of growing facial mesenchyme in normal and disease models.