Intrinsic Mechanisms are Responsible for Embryonic Midfacial Narrowing

Objectives: Our study investigated the extrinsic versus intrinsic factors contributing to midline narrowing and tested the role of the ROCK, a mediator of the planar cell polarity pathway. The amniote face (mammals, reptile) undergoes conserved shape changes during embryonic development. One of the most rapid events is the relocation of the nasal pits from the side of the head to the midline. This narrowing of the midline is due to merging of the medial nasal processes but the mechanism is unknown. We hypothesized that the non-canonical WNT pathway (JNK) may be controlling facial narrowing.
Methods: In-vivo: chicken faces were analyzed in 3D using the Optical Projection Scanner followed by 3D Geometric morphometrics. In vitro- the frontonasal mass was dissected from stage 25 chicken embryos and grown for 48h in DMEM/F12 media with/without the eyes and brain. Another set was treated with a ROCK inhibitor (2.5 µM Y27632). Organ cultures photographed every 24h. Another set was used for time-lapse confocal microscopy for 6-12h.
Results: In vivo data showed that the shape of the frontonasal mass narrows in the mediolateral axis while extending in the perpendicular axes. In vitro organ cultures showed that significant narrowing occurred independent of whether eyes the brain was included. Thus, intrinsic factors are important. The ROCK inhibitor completely blocked narrowing. Time-lapse cell tracking of labeled cells showed that the ROCKi caused cells to move dorsally, leading to flattening and widening of the culture.
Conclusions: Our study suggests that convergent extension is taking place in the frontonasal mass and the activity of the planar cell polarity pathway is required. Live cell imaging techniques open new avenues to observe growing facial mesenchyme.