Hmx1 Function In Lateral Facial Development

Thesis (Master's)--University of Washington, 2016-08

The oculoauricular syndrome (OAS) in humans, the “dumbo” and “misplaced ears” strains in mice, the “dumbo” strain in rats, and the “crop ear” phenotype in cattle, all converge on a single gene - Hmx1. It encodes a homeodomain transcription factor expressed in the developing eyes, peripheral nervous system, and posterior aspects of the first and second branchial arches (BA1 and BA2) which give rise to lateral craniofacial structures including the external ear. Current published literature on lateral craniofacial development and its associated anomalies is scarce. To enhance the knowledge in this field, we used high-resolution imaging tools and performed 3D morphological studies on staged mouse embryos and also on postnatal day 28 (adult) mice to characterize in detail the phenotype of the dumbo mutants. In addition to the rotated low-set position of the dumbo ear, a novel ear tag was found at the lower back of the pinna when dumbo mice were examined in a pure C57Bl6/N background. This structure could be identified as early as embryonic day (E)12. Abnormalities were also uncovered in adult dumbo mice: fusion in the squamosal and parietomastoid sutures, shape changes in the cranium and mandible, paraoccipital process hypoplasia, auriculofacial nerve deficits, and a lack of response to an air-blowing test. These all suggest Hmx1 is required for the development of lateral facial structures and proper neuromuscular reflex. Unlike the Hmx1 coding region mutations which were found responsible for the mouse phenotype and human OAS, our study with the dumbo rat strain showed that a 5.7 kb deletion, ~80 kb distal to the Hmx1 locus, is responsible for the rat dumbo phenotype. Transient transgenesis tests demonstrate that this deleted non-coding sequence encompasses a 594 bp Evolutionarily Conserved Region (ECR) that functions as an Hmx1 tissue-specific enhancer regulating the development of specific lateral and frontonasal mesenchyme. Future fate-mapping studies using this tissue-specific ECR will ultimately determine the specific craniofacial tissue types derived from the embryonic Hmx1-expressing mesenchyme. Studies of the Hmx1 regulatory network could provide a defined blueprint to interpret the genetic contribution to pinna morphogenesis and other birth defects associated with the lateral facial structures.