Characterization of the function of the Otx-2 gene in early mammalian development

grantor: University of TorontoHow the anterior region of the vertebrate embryo is specified is still an unsolved question in early pattern formation. In this thesis, I have undertaken a study of the roles of a homeobox gene, 'Otx-2', in the early murine development, with particular reference to its role in anterior patterning. 'Otx-2', one of the two mammalian Otx genes, related to the 'Drosophila otd' gene, has been cloned and characterized. It encodes a protein containing a 60 amino acid homeodomain motif with only two amino acid differences from the sequence of 'Drosophila' Otd. 'Otx-2' is one of the earliest genes showing restricted anterior expression domains. During early embryogenesis, 'Otx-2' is first expressed in the entire epiblast which gives rise to the embryo proper, then is gradually restricted to the anterior region of the embryo and finally to the presumptive forebrain and midbrain. This expression profile suggests that 'Otx-2' may have multiple roles in the early development and anterior patterning of the embryo. These studies provide fundamental background information for further functional analysis. Functional analysis of 'Otx-2' was performed by a gene targeting approach. A loss-of-function mutation of 'Otx-2' leads to loss of forebrain, midbrain and anterior hindbrain, suggesting the critical role of 'Otx-2' in head formation. In addition, homozygous ' Otx-2' mutant embryos also show early gastrulation defects and defects in the prechordal mesoderm and notochord precursors. These defects are consistent with multiple roles for 'Otx-2' in gastrulation and the patterning of the rostral brain in mice. To further dissect the function of 'Otx-2' in anterior patterning, a double heterozygous mutant was made with 'HNF-3'ß, a member of the winged-helix transcription factor family. Generation of ' Otx-2'+/-; 'HNF'-3ß+/- double heterozygous mutants demonstrates a new lethal phenotype in mice. The phenotype is characterized by varying degrees of holoprosencephaly, cyclopia with proboscis-like structures, suggesting the genetic interaction of the two genes in anterior midline patterning. Co-expression of 'Otx-2' and 'HNF-3'ß in the anterior midline of the embryo further supported such interactions. Furthermore, the loss of 'Shh' expression, a secreted protein, in the anterior region of 'Otx-2'+/-;'HNF-3'ß+/- embryos indicates that both 'Otx-2' and ' HNF-3'ß are involved in regulating the 'Shh' signalling pathway in the anterior midline. This study also illustrates the way in which dosage-dependent effects of transcription factors can reveal novel genetic interactions and help define genetic pathways.Ph.D