Our laboratory uses genetic and developmental approaches, exploiting the mouse as a model system, to study skeletal patterning and morphogenesis during development. To this end, we are performing a phenotype- based forward genetic screen by ethylnitrosourea (ENU) mutagenesis in the mouse to uncover novel genes important in mammalian craniofacial development. Within the past two years, the screen produced four mutant mouse lines with skeletal defects. Two of these lines exhibit remarkable craniofacial malformations. Specifically, mutants from line 04/014 develop a short mandible, a hypoplastic or absent hyoid, and rudimentary neck cartilages. Also, their palate is cleft and the basisphenoid is fused to the basioccipital bone. Additionally, 04/014 mutants exhibit low-set and hypoplastic ear pinnae and their middle ear ossicles are severely affected. Finally, the cartilage primordium of the petrous part of the temporal bone is absent, leaving a hollow space (therefore this line will be defined hereafter as """"""""Hollow ear"""""""", or Hol), and the Otic Capsule (part of the lateral chondrocranium, containing the developing inner ear apparatus) is hypoplastic and dysmorphic. Overall, the Hol craniofacial mutation phenocopies the Tbx1 homozygous mutation in the mouse, which models DiGeorge Syndrome (DGS). We propose that the Hol mutation disrupts a gene essential for vertebrate craniofacial and ear development. Indeed, by exploiting high-resolution genetic mapping, we discounted that the Hol mutation maps to either the Tbx1 or Crkl loci on chromosome 16. Furthermore, we mapped the Hol mutant gene to an interval of approximately 8 Mb on mouse chromosome 11, by exploiting novel mapping technology based on whole genome scanning using single nucleotide polymorphisms (SNP) panels. Finally, since the last amended submission of this proposal, we have further narrowed the Hol-bearing interval to approximately 3.9 Mb, by high-resolution mapping and polymorphic markers. We hypothesize that the Hol gene acts in the Tbx1 path- way in craniofacial development, but Hol is not Tbx1 or Crkl. We plan to uncover the molecular basis of this mutant phenotype through the following specific aims: 1) Identify Hol candidate genes, by performing further high-resolution mapping and microarray analysis for """"""""gene finding"""""""";and 2) Identify the Hol gene, by conducting analysis of candidate genes and identification of the Hol molecular lesion by sequencing of candidate genes. Completion of these studies will advance our understanding of the genetic regulation of craniofacial patterning and morphogenesis, as well as lead to the discovery of a new gene that likely acts in the Tbx1 pathway. Under a broader perspective, this work will have an impact on the pathogenesis of human congenital disorders that affect craniofacial and ear development and function, such as DiGeorge Syndrome (DGS).
Knowledge of the patterning and morphogenesis of craniofacial and ear structures and of the genes implicated in their developmental processes is still elementary. Completion of the studies proposed here will represent a step forward in our understanding of the genetic regulation of patterning and morphogenesis of the vertebrate cranium. Furthermore, this work will uncover a novel gene that likely acts in a genetic pathway together with Tbx1 and Crkl and is required for critical craniofacial developmental processes. Under a broader perspective, these studies will have an impact on our knowledge of the pathogenesis of human congenital disorders that affect normal craniofacial and ear development and function, in particular with regard to the malformations of DiGeorge Syndrome.
