Congenital anomalies affect 1% of live births, with one-third of these displaying craniofacial abnormalities. Great advances have been made in our understanding of the genetic causes of many of these conditions, but lack of understanding of the underlying developmental, cellular, and molecular mechanisms continues to stymie personalized medicine approaches to treat these conditions. Craniofrontonasal syndrome (CFNS) is an X-linked condition caused by mutations in the EFNB1 gene that affects craniofacial and thoracic skeleton, and neurological development. Complete loss of EFNB1 function results in hypertelorism and cleft palate, but mosaic loss of EFNB1 in only some cells, has more severe consequences, resulting also in midfacial hypoplasia, coronal craniosynostosis, limb abnormalities, and agenesis of the corpus callosum. Mouse Efnb1 mutants effectively model CFNS, exhibiting most of the same phenotypes. Efnb1 encodes Ephrin-B1 a transmembrane signaling molecule that binds to EphB receptor tyrosine kinases to regulate cell position. In Efnb1+/- heterozygous females, X chromosome inactivation leads to mosaicism for Ephrin-B1 function and subsequent sorting-out of Ephrin-B1-expressing cells from Ephrin-B1 mutant cells. This self-organizing capacity is a general feature of Eph/Ephrin signaling in many different tissues. In craniofacial development, we hypothesize that Ephrin-B1 plays a critical role in the organization of the craniofacial mesenchyme, though how this occurs, and how it affects craniofacial shape, remains unknown. It is therefore our goal to understand how Ephrin-B1 regulates normal craniofacial morphogenesis at the developmental, cellular and molecular scales. First, to understand how Ephrin-B1 regulates changes in shape of specific craniofacial embryonic primordia, we will utilize micro-computed tomography scanning to perform landmark-based morphometric analysis of mouse embryos lacking Ephrin-B1 in specific structures. Using a similar approach, we will evaluate the relative involvement of three receptors EphB1, EphB2 and EphB3 in craniofacial morphogenesis. Second, we will utilize new tools to study the distribution, shape, density, and polarity of the craniofacial mesenchyme in normal and Efnb1 mutant embryos. Finally, we will harness CRISPR genome editing technology to perform a functional screen of candidate genes to identify the signaling pathways downstream of Ephrin-B1/EphB signaling. This work will provide new insights into the mechanisms by which Ephrin-B1 normally controls mesenchymal cell behaviors in craniofacial morphogenesis, and determine how its loss results in pathological changes in craniofacial shape. These studies will therefore provide foundational knowledge toward our long- term goal of developing improved therapies for the treatment of individuals with craniofacial anomalies and an improved understanding of the fundamental processes of craniofacial morphogenesis. The underlying cellular and molecular mechanisms may also be relevant to any of the numerous contexts where Eph/Ephrin signaling is important in development and disease.
This proposal focuses on craniofrontonasal syndrome (CFNS), an X-linked disease that affects multiple aspects of craniofacial development and is caused by mutations in the EFNB1 gene. This proposal will define the embryonic contributions to the broader CFNS pathology and determine how EPHRIN-B1 signaling pathways regulate mesenchymal cell behaviors to drive these phenotypes. This work will provide insight into the developmental functions of EFNB1, and build a foundation of understanding for the future design of preventative or therapeutic strategies for CFNS.
