PROJECT SUMMARY The lower jaw evolved as a structure composed of many bones the largest of which, the dentary, persists as a single lower jawbone in modern mammals, and is referred to as the mandible in humans. Mandibular disorders, often resulting in small jaws, are among the most common human birth defects. These disorders can dramatically affect quality of life, and are often associated, or compound problems with airway obstruction, speech, and feeding. During embryogenesis, development of the mandible is preceded by and associated with a tubular cartilage rod called Meckel's cartilage (MC) and anomalies of MC have been associated with mandibular disorders. Development of MC in modern mammals is complex: the anterior part contributes to the formation of the mandibular symphysis, its posterior part forms cartilages that mineralize endochondrally to form two middle ear bones, and the posterior half of the middle region forms ligaments. Less is known about the anterior half of the middle region of MC, which is transient, present during a small window of embryonic development before it disappears. Established assessments describe MC as a template for the formation of the mandible, but evidence of this is lacking and little is known of the relationship between the midportion of MC and mandibular mineralization, size, and shape or the processes of MC growth in length, perichondrial ossification, and disappearance of MC. We present data demonstrating that MC does not serve as a template in the way cartilaginous models function in endochondral ossification and hypothesize a new role for the mid portion of MC in determining mandibular length, mineralization of the perichondrium, mineralization of the mandible, and its disappearance. Because our findings challenge the traditional role of MC, we have designed this project to validate the developmental events that take place as the midportion of MC disappears and the mandible forms through a detailed analysis of four processes: 1) initiation and growth in length of MC; 2) mineralization of MC perichondrium; 3) mineralization of the mandible; and 4) disappearance of MC. Our approach is based on knowledge we have gained through preliminary investigations of these processes occurring at different times along the length of MC, and spanning the buccal to lingual aspects of the interior of MC. We couple 3D imaging with tissue and cellular analyses of embryonic mutant Sox9flox/flox;Col2a1-CreERT, and Sox9flox/flox mice to precisely define the changing cellular dynamics of the lower jaw in developmental time and anatomical space. These data are used in turn to inform our RNA-seq analyses of the developing MC and mandible directed at recovering the underlying transcriptome by differential gene expression, pathway, and network analyses. We plan cell lineage tracing experiments to determine the fate of cells from the intermediate region of MC, those that initiate MC perichondrial mineralization, and mandible mineralization. Our integrative approach is designed to bring new understanding to lower jaw development and open novel research areas to advance strategies for bone repair, regeneration, and prevention in mandibular disease.

PROJECT NARRATIVE Mandibular anomalies are common birth defects that require comprehensive, sometimes repetitive corrective surgeries to manage individual cases, often placing a heavy financial and emotional burden on patients and their families. Development of therapeutic efforts requires a thorough understanding of mandibular development and the timing of events in dysmorphogenesis. We offer innovative approaches for determining the molecular, cellular, and developmental bases of morphogenesis of the mandible and its cartilaginous precursor, Meckel’s cartilage, in normal individuals and in craniofacial conditions with a small mandible.