UCSF

Objectives: Craniofacial malformations are among the most common birth defects, affecting ~1 in every 700 live births. However, our understanding of the mechanisms that result in these diseases is limited. Many cases have been associated with genetic predispositions that have allowed us to attempt to investigate the underlying mechanisms for these diseases. Sonic Hedgehog (SHH) signaling pathway has undergone significant investigation due to the involvement of Shh in the development of the midface. In humans, deletion of a single copy of Shh is associated with a spectrum of phenotypes comprising Holoprosencephaly (HPE), ranging from mild hypotelorism and midfacial hypoplasia to cyclopia indicates that diseases like HPE are multifactorial. Although our long-term goal is to discover the underlying mechanisms that cause these diseases, we must first understand the normal progression of development before trying to understand the abnormal. Therefore, the goal of this study was to determine how Shh expression and face shape relate with each other during normal development.

Methods: We collected wild-type chicken embryos at 72 hrs, 96 hrs, and 120 hours of incubation. Chickens infected with RCAS-wnt3a at 72 hrs of incubation were also included in our sample. We used in situ hybridization to identify Shh expression domains and 2D geometric morphometrics to quantify changes in shape in Shh expression domains and face shape. We performed Principal Components Analysis (PCA) as well as multivariable regression analyses of Shh expression shape and face shape on somite number and centroid size. We used Partial Least Squares (PLS) to evaluate covariation in shape between Shh expression domains and facial shape.

Results: Changes in Shh expression shape and face shape are dependent on developmental time. As the embryos progressed in development, there were significant changes in both Shh expression shape and face shape. While the overall size of the embryo grew, both the Shh expression shape and face shape constricted. More specifically, Shh expression shape tapered into a narrow ¬V-shaped band in the ectoderm of the stomodeum while face shape constricted as a result of the nasal pits growing closer together while the mouth became smaller. Our PLS regression identified that the changes in Shh expression and face shape are correlative in which Shh expression shape is associated with face shape at specific timepoints during development.

Conclusion: Changes in Shh expression shape correlate with changes in face shape. This suggests that Shh expression shape may serve as a predictor for face shape during embryonic growth. Although we are unable to determine if Shh is directly responsible for the observed changes in face shape, this potential predictive relationship could be valuable for future studies to identify when and how disease progression initiates.