The goal of this project is to identify microRNAs (miRNAs) involved in vertebrate mid-face development and to determine their function in this process. Our hypothesis is that a limited number of miRNAs play crucial roles in the patterning, proliferation and differentiation of the mid-face through precise spatial and temporal expression of specific protein-coding genes. Further, we hypothesize that both loss- and gain-of-function of these miRNAs leads to mid-facial and orofacial defects/clefting. In humans, defects in mid-facial development, including cleft lip/palate, account for the largest number of birth defects annually. Alone, over 400 syndromes result in cleft lip and palate, which has an occurrence rate of 1 in 800 live births. Our laboratories have a long-standing interest in determining the gene regulatory networks that control normal orofacial development, many of which are causative in animal models of mid-facial clefting. In our Preliminary Data, we show that microarray technology can generate comprehensive miRNA expression profiles and that bioinformatic analysis can define miRNAs whose expression patterns are conserved across vertebrates. In this proposal, we will investigate the identity and function of miRNAs expressed in the developing mid-face in three Specific Aims.
In Aim 1, we will determine and validate the temporal and spatial expression patterns of miRNAs in the developing mouse maxillary/frontonasal prominences using miRNA microarray technology.
In Aim 2, we will determine the in situ expression patterns of identified miRNAs in both mouse and zebrafish embryos to define those that show a conserved pattern of expression.
In Aim 3, we will use zebrafish to determine the regulatory function and morphogenetic mode of action of the miRNAs by loss- and gain-of-function analysis. We propose using both mouse and zebrafish systems because their complementary well-established experimental and genetic methods make a detailed analysis of this process feasible. By defining miRNAs involved in mouse mid-facial development and then quickly and efficiently determining miRNA function in zebrafish, we will be able to elucidate the miRNAs involved in vertebrate mid-facial development. This is crucial to further our understanding of genetic events leading to human mid-facial birth defect syndromes.
Mid-facial birth defects represent a significant social and medical problem throughout the world. By defining the miRNAs involved in mid-facial development, we expect to uncover novel genetic regulatory mechanisms involved in normal and abnormal mid-face development. Our characterization of miRNAs in the mid-face will provide potential targets for the development of novel therapeutics, with the goal of repairing and preventing mid-facial human congenital defects.
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