| Transcription factor AP-2α is required for appropriate craniofacial morphogenesis. It is expressed in the cranial neural crest and developing facial prominences throughout embryogenesis. Targeted disruption of the AP-2α gene (AP-2KO) leads to severe midline facial clefting, while chimeric animals composed of wild-type and AP-2alpha-null cells exhibit cleft lip and/or palate. AP-2α, therefore, is essential for proper development of tissues derived from the embryonic frontonasal prominence (FNP). Its specific roles in these processes, however, remain elusive. In order to understand how AP-2α functions in craniofacial development, two distinct approaches have been taken.; The first approach utilizes conditional mutagenesis to eliminate AP-2α specifically from craniofacial tissues (Chapter 2). This study describes the generation and characterization of a FNP- and limb bud-specific CRE recombinase allele (AP-2CRE), driven by AP-2α promoter and enhancer elements. AP-2CRE was then used to generate a new mutant (FKO) in which AP-2α is disrupted in the FNP and limbs. FKO mice have slightly shortened snouts and wide-set eyes that are obvious approximately 15 days after birth. Examination of craniofacial FNP derivatives from FKO mice reveals defects in nasal bones and cartilage, facial sutures, and the nasal mucosa. This is the first evidence that AP-2α functions in postnatal craniofacial morphogenesis.; A second line of experiments was initiated in order to identify potential AP-2α target genes (Chapter 3). To determine if target genes could be isolated directly from AP-2KO embryos, tissue microdissection was coupled with microarray hybridization. Wild-type and AP-2KO FNP tissues were processed for screening on cDNA arrays and Affymetrix gene chips. Dickkopf-1 (dkk-1) and tumor protein D53 were identified in the screen and display FNP-specific delays in the onset of expression. dkk-1 is a Wnt antagonist, and targeted disruption results in a loss of structures anterior to the midbrain. D53 is found to be upregulated in cancer cells, but its expression in embryonic development was previously unreported. These studies have shown that the identification of putative AP-2α targets is possible with a microdissection approach, and have provided insight into the potential in vivo roles of AP-2α in facial development. |
