PRS AAPS Oral Proofs 2016
Lucie Rochard, PhD,* Stefanie Monica, BS,† Irving Ling, MD, PhD,* Yawei Kong, PhD,* Eric C. Liao, MD, PhD*
From the *Harvard Medical School, Boston, Mass.; and †University of California Berkeley, Berkeley, Calif.
PURPOSE: Genetic regulation of cells to organize into higher ordered structures is a central question of craniofacial biology. Wnt signaling is a key pathway that regulates tissue morphogenesis and convergence extension (CE) cell movements. We hypothesize that palate formation requires Wnt regulation of CE, and when this process is disrupted, orofacial clefts result.
METHODS:CRISPR-Cas gene editing was used to generate mutants representing components of the Wnt pathway: Wls (chaperon for Wnt secretion), Wnt9a, Wnt5b (ligands), Frzb, and Gpc4 (receptors). Double mutants were generated to determine relative contribution of each gene to palatogenesis. Detailed cellular and molecular analyses were performed.
RESULTS: Gene expression analysis localized Wnt signal originating from oropharyngeal epithelium, regulating juxtaposed palate chondrocytes expressing receptors frzb and gpc4. CE behaviors of cell intercalation and directional proliferation were revealed by multispectral clonal analysis in the mutants. Genetic studies showed that wls and gpc4 are required to impart anteroposterior and dorsoventral cues, wnt5b is required for anteroposterior extension, wnt9a is required for dorsoventral extension, and frzb regulates cell orientation.
CONCLUSIONS: Systematic genetic analysis revealed the central role of the oral epithelium to generate the Wnt gradient, imparting positional cues to developing chondrocytes. Different Wnt genes contribute differently to axis cues that regulate chondrocyte behavior. Elucidating Wnt regulated cell behavior is a key to understanding normal palate development and cleft pathogenesis when this process is perturbed.