Frontonasal dysplasia (FND), also known as median cleft face syndrome, is a major class of craniofacial birth defects that profoundly impact the form and function of the face. FND patients require multiple corrective surgeries and often suffer life-long impairment. Whilst most FND cases occur sporadically with unknown etiology, loss-of-function mutations in each of the three ALX family genes, ALX1, ALX3, and ALX4, have been identified as the genetic causes for autosomal recessive FND, with disruption of ALX1 associated with severe facial clefting and extreme microphthalmia in FND3 patients while mutations in ALX3 and ALX4 resulted in milder but clinically distinctive frontonasal malformations. Little is known about how ALX transcription factors regulate craniofacial development, and the overall molecular mechanism controlling frontonasal development is poorly understood. In preliminary studies, we have generated Alx1 mutant mice using CRISPR-mediated genome editing and found that they recapitulated the FND3 phenotypes, including reduced frontonasal bones and cartilages, cleft palate, and microphthalmia. We found that Alx1-/- embryos exhibited ectopic neuroglial differentiation and reduction in ectomesenchymal gene expression in the frontonasal prominence. Moreover, Alx1-/-Alx4-/- double mutant mouse embryos exhibited increased ectopic cranial ganglia and much severer frontonasal deficiency than Alx1-/- mutants. Previous studies in multiple animal model systems revealed that the Twist1 transcription factor, whose expression is activated in cranial neural crest cells at the onset of migration, is critical for ectomesenchyme specification. Remarkably, while the molecular mechanism acting downstream of Twist1 in promoting ectomesenchymal fate is still unclear, Twist1-/- mouse embryos failed to activate the expression of all three Alx genes in cranial neural crest cells. Our finding of ectopic neuroglial differentiation in the frontonasal regions of Alx1-/- and Alx1-/-Alx4-/- embryos suggests that Twist1 and the ALX transcription factors act in the same molecular network to regulate cranial neural crest fate determination between the ectomesenchymal and neuroglial lineages.
The specific aims of this research project are to determine the cellular and molecular mechanisms mediating ALX transcription factor function in frontonasal development and to unravel and reconstruct the gene regulatory network consisting of Twist1 and ALX transcription factors regulating cranial neural crest differentiation. Results from these studies will fill a longstanding critical gap in craniofacial developmental biology and lead to new improvements in molecular diagnosis and treatment/care of a large number of craniofacial disorders.
Frontonasal dysplasia (FND), also known as median cleft face syndrome, is a large class of craniofacial birth defects that profoundly impact the form and function of the face, nose, and mouth. FND patients require multiple corrective surgeries and often suffer life-long impairment. Using novel mouse models and innovative research approaches, this project will uncover the underlying developmental and molecular mechanisms and lead to significant improvement in molecular diagnosis, genetic counseling, and treatment of patients with FND and related craniofacial disorders.