Project Grant R15DE033850

MOLECULAR AND CELLULAR MECHANISMS UNDERLYING FGF8-DOSAGE MEDIATED DEFECTS IN JAW SIZE AND SYMMETRY - PROJECT SUMMARY CRANIOFACIAL MALFORMATIONS ARE AMONG THE MOST COMMON HUMAN BIRTH DEFECTS. TISSUES OF THE HEAD AND NECK DERIVE FROM THE PHARYNGEAL ARCHES (PAS). PAS ARE TRANSIENT DEVELOPMENTAL STRUCTURES FORMED BY MESENCHYMAL POPULATIONS, THE NEURAL CREST (NC) AND MESODERM, THAT MIGRATE IN BETWEEN EPITHELIAL LAYERS, THE SURFACE ECTODERM AND FOREGUT ENDODERM. OUR WORK FOCUSES ON THE JAW SKELETON, WHICH DERIVES FROM NC OF THE FIRST PHARYNGEAL ARCH (PA1). PHARYNGEAL EPITHELIA HAVE AT LEAST 3 CRITICAL ROLES IN JAW DEVELOPMENT: 1) SEGMENTING PA1 FROM PA2, 2) PROVIDING SIGNALS THAT SUPPORT PATTERNING AND PROLIFERATION OF PA1 MESENCHYME, AND 3) SUBSEQUENTLY DIFFERENTIATING INTO TISSUE DERIVATIVES OF THE EAR. TO BETTER UNDERSTAND HOW PHARYNGEAL EPITHELIA REGULATE JAW DEVELOPMENT, WE INVESTIGATE THE DEVELOPMENT AND MORPHOGENESIS OF THE FIRST PHARYNGEAL POUCH (PP1) AND FIRST PHARYNGEAL CLEFT (PC1), WHICH SEGMENT PA1 AND PA2. THE SIGNALING FACTOR, FGF8, IS EXPRESSED IN BOTH PP1 AND PC1. OUR PREVIOUS WORK HAS SHOWN THAT JAW DEVELOPMENT IS SENSITIVE TO FGF8 DOSAGE AND EXHIBITS DIRECTIONAL ASYMMETRY, WITH THE LEFT SIDE BEING MORE SEVERELY AFFECTED THAN THE RIGHT. SIMILAR DIRECTIONAL ASYMMETRY TRENDS ARE OBSERVED IN HUMAN CRANIOFACIAL MALFORMATIONS. WE ALSO FOUND THAT FGF8-MEDIATED DEFECTS OF THE JAW SKELETON ARE ASSOCIATED WITH 1) ALTERATIONS TO PATTERNING OF PA1 NC AND 2) MALFORMATIONS IN BOTH PP1 AND PC1. OUR DATA INDICATE THAT FGF8 IS CRITICAL TO PP1 AND PC1 DEVELOPMENT, BUT THE SPECIFIC CELLULAR MECHANISMS MEDIATED BY FGF8 ARE UNKNOWN. IN OUR PREVIOUS WORK, WE DEFINED 4 STAGES OF PP1 AND PC1 MORPHOGENESIS, INCLUDING AN EXTENDED PERIOD OF CONTACT BETWEEN PP1 AND PC1, WHICH IS DISRUPTED IN FGF8 MUTANTS. THE INTERACTION BETWEEN PP1 AND PC1 IS TRANSIENT, AS THE TWO EPITHELIA LATER SEPARATE TO COMPLETE THEIR DIFFERENTIATION. WE HYPOTHESIZE THAT FGF8 IS REQUIRED TO MEDIATE REGIONAL CELL IDENTITY IN PP1 AND PC1 TO FORM A BOUNDARY AND MEDIATE EPITHELIAL EXTENSION. IN AIM 1, WE WILL FURTHER EXPLORE HOW FGF8 DOSAGE IMPACTS EPITHELIAL CELL BIOLOGY. A SECOND ASPECT OF OUR PREVIOUS WORK THAT REMAINS UNEXPLAINED IS THE MECHANISM UNDERLYING DIRECTIONAL ASYMMETRY IN FGF8 MUTANT JAWS. IN AIM 2, WE WILL FURTHER EXPLORE HOW FGF8 DOSAGE CONTRIBUTES TO DIRECTIONAL ASYMMETRY IN JAW DEVELOPMENT. WE HYPOTHESIZE THAT PROGENITORS OF THE HEART FIELD MIGRATING THROUGH PA1 PROVIDE SMALL AMOUNTS OF FGF8 THAT BUFFER PA1 DEVELOPMENT WHEN FGF8 DOSAGE IS LOW. FINALLY, IN AIM 3, WE WILL BEGIN TO EXPLORE HOW FGF8 DOSAGE IS FURTHER MODULATED THROUGH SPLICE VARIANTS. FGF8 MRNA EXHIBITS SPLICE VARIANTS. THE TWO MOST COMMON PROTEIN ISOFORMS, FGF8A AND FGF8B, HAVE DISTINCT ERK SIGNALING ACTIVITY. THESE DATA SUGGEST THAT MRNA SPLICING MAY MODULATE FGF8 DOSAGE BEYOND THE CIS- REGULATORY MECHANISMS WE HAVE PREVIOUSLY STUDIED. WE WILL USE BOTH IN VIVO AND IN VITRO MODELS TO TEST OUR AIMS. THIS WORK HAS IMPLICATIONS FOR CRANIOFACIAL DISEASE SYNDROMES THAT ALSO INCLUDE HEART DEFECTS, SUCH AS CHARGE AND DIGEORGE (22Q11 DELETION) SYNDROMES. THESE SYNDROMES ALSO EXHIBIT ASYMMETRY IN JAW AND EAR DEFECTS.