Empirical evidence exists for a genetic hierarchy of sox2/sox3>atonal>and brnSc in the neural induction and patterning of the otic placode (sox2/sox3) of and the subsequent neural differentiation of mechanosensory structures from this primordium (atonal and brnSc). A host of syndromic human disorders are associated with these genes. Prominent syndromes associated with sox2 mutations encompass anopthalmia and microopthalmia whereas mutations in brnSc mutations are associated with progressive deafness. However, the location of the core mutations in many of these and other neurodevelopmental disorders map outside of the reading frame of the gene. Thus, alterations in transcriptional regulation likely underlie several syndromic hearing and deafness disorders. Additionally, the enhancer elements of all the genes in question (with the exception of Sox2) are unknown or poorly classified. While a definitive hierarchy exists between the aforementioned genes, the existence of direct or indirect interactions between genes is unknown. The to!2 transgenic system I propose here in conjunction with the power of comparative genomics will identify the enhancer elements responsible for the expression of each of the aforementioned genes in the zebrafish lateral line system (which serves as an excellent completely external model of hair cell development in the inner ear) as well as establish an indirect or direct relationship for all genes in the cascade. Identification of functional enhancers is likely to elucidate enhancer elements with associated deafness or hearing disorder syndromes.
Aim1 : Characterize all potential enhancers of the sox2/sox3 genes, atohla, and brnSc genes within roughly a megabase window of each gene.
Aim2 : In order to test the domains critical to the enhancer activity for each identified element element, I will perform a series of deletion constructs to determine which regions and transcription factor binding sites are essential to enhancer activity.
Aim3 : Given the success of aims1-2,1 would like to extend this method broadly to transcription and growth factors involved in the formation of the lateral line. Public Health Statement Mutations involved in hearing often correspond to sequences controlling the expression of a gene rather than changes inherent to the gene sequence. By comparing the sequences around shared genes involved in inner ear formation across many vertebrates, I will discover which non-genie sequences are critical to hearing using the zebrafish lateral line system (which detects vibration) as a model for the human inner ear. ? ? ? ?
