Characterization of the Essential Role of the miR-200 family of microRNAs in Mucociliogenesis
- Author(s): Cisson, Jennifer Lauren
- Advisor(s): He, Lin
- et al.
MicroRNAs are small, noncoding RNAs that play essential roles in regulation of a variety of processes including development, cellular feedback mechanisms, and reproductive biology. The studies that follow focus on a particularly pleiotropic set of miRNAs: the miR-200 family. For example, the miR-200 family is known to be crucial for differentiation and maintenance of specialized epithelial cell types, which corresponds to its role in regulation of Epithelial-Mesenchymal Transition (EMT).1–4 miR-200 consists of five homologous members, divided between two distinct genomic loci and can be further segregated into two subfamilies based on seed sequence homology: miR-200a/141 and miR-200b/200c/429. In an effort to discern the importance of the miR-200 family of miRNAs, we acquired and intercrossed mir-200b/a/429 (“200ab”) and mir-200c/141 (“200c”) heterozygotes to breed single and double knockout mice (a generous gift from the Bradley Lab at the Sanger Institute).5 I discovered a striking postnatal lethality in mir-200ab/c double knockout (DKO) progeny, whereby neonates survive less than 16 hours post parturition. In an effort to characterize the lethal effect of losing miR-200, I performed behavioral studies, expression profiling, histopathological analysis, and electron microscopy. Interestingly, DKO pups exhibit no milk spot, and develop labored breathing, along with pale, sticky skin within hours after birth. As expected, epithelial tissues such as the lungs and gastrointestinal tract exhibit the highest expression level of miR-200 between E18.5 and P0. Although tissue architecture appears relatively normal in DKO epithelial tissues immediately after birth, I observed rapid accumulation of debris and mucus in the large and small airways that correlates with cyanosis. Intriguingly, the phenotypic changes in the lungs, gut, and skin of DKO pups are comparable to human symptoms of cystic fibrosis and asthma, which may provide novel insights into these diseases, where current mouse models fail.6,7 We have acquired a mir-200c/141: LacZ reporter (a generous gift from the McManus Lab, UCSF), and used double immunofluorescence (IF), FACS, and single cell qPCR to determine the cell population expressing miR-200 is largely motile ciliated cells (MCCs). In addition, we have found that miR-200 is highly enriched in MCCs during frog development in published datasets.8 Finally, in single (200ab) knockout males, I observed an infertility defect that correlates with mislocalized flagella – a structure related to the cilia in MCCs. Future studies include further characterizing the mucociliary defect, and identifying miR-200 targets. Overall, I have shown that miR-200 is absolutely essential for the proper function and maintenance of the mucociliary epithelium lining the airways. Loss of miR-200 leads to excessive mucus secretion into the airway lumen, and correspondingly, respiratory distress culminating in death.