ABSTRACT
RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2+ cells increased after knockdown, whereas P63/SOX2+ cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK's role during lung development.
Subject(s)
Cell Differentiation , Cilia/physiology , Gene Expression Regulation, Developmental , Lung/embryology , Lung/metabolism , RNA-Binding Proteins/metabolism , Animals , Female , Protein Isoforms , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Congenital diaphragmatic hernia (CDH) is a developmental birth defect consisting of a diaphragmatic defect and abnormal lung development. CDH complicates 2.3-2.8 per 10,000 live births. Despite efforts to standardize clinical practice, management of CDH remains challenging. Frequent re-evaluation of clinical practices in CDH reveals that management of CDH is evolving from one of postnatal stabilization to prenatal optimization. Translational research reveals promising avenues for in utero therapeutic intervention, including fetoscopic endoluminal tracheal occlusion. These remain highly experimental and demand improved antenatal diagnostics. Timely diagnosis of CDH and identification of severely affected fetuses allow time for delivery planning or in utero therapeutics. Optimal perinatal care and surgical treatment strategies are highly debated. Improved CDH mortality rates have placed increased emphasis on identifying and monitoring the long-term sequelae of disease throughout childhood and into adulthood. We review the current management strategies for CDH, highlighting where progress has been made, and where future developments have the potential to revolutionize care in this vulnerable patient population.
Subject(s)
Disease Management , Fetus/diagnostic imaging , Hernias, Diaphragmatic, Congenital/surgery , Practice Guidelines as Topic , Prenatal Diagnosis/methods , Female , Fetoscopy/methods , Follow-Up Studies , Hernias, Diaphragmatic, Congenital/diagnosis , Humans , Infant, Newborn , Pregnancy , Time FactorsABSTRACT
PURPOSE: Epigenetic factors are involved in the pathogenesis of congenital diaphragmatic hernia (CDH). Circular RNAs (circRNAs) are epigenetic regulators amenable to biomarker profiling. Here, we aimed to develop a liquid biopsy protocol to detect pathognomonic circRNA changes in biofluids. METHODS: Our protocol is adapted from the existing BaseScope™ in situ hybridization technique. Rat biofluids were fixed in a gelatin-coated 96-well plate with formalin. Probes were designed to target circRNAs with significant fold change in nitrofen-induced CDH. FastRED fluorescence was assessed using a plate reader and confirmed with confocal microscopy. We tested maternal serum and amniotic fluid samples from control and nitrofen-treated rats. RESULTS: We detected circRNAs in rat serum and amniotic fluid from control and CDH (nitrofen-treated) rats using fluorescent readout. CircRNA signal was observed in fixed biofluids as fluorescent punctate foci under confocal laser scanning microscopy. This was confirmed by comparison to BaseScope™ lung tissue sections. Signal was concentration dependent and DNase resistant. CONCLUSION: We successfully adapted BaseScope™ to detect circRNAs in rat biofluids: serum and amniotic fluid. We detected signal from probes targeted to circRNAs that are dysregulated in rat CDH. This work establishes the preliminary feasibility of circRNA detection in prenatal diagnostics.
