Congenital diaphragmatic hernia (CDH) affects 1 in 2500 live births with mortality rates in newborns ranging from 10-40%, depending on factors such as severity. CDH involves the herniation of the abdominal organs into the chest cavity through a hole in the diaphragm and results in decreased lung growth, or pulmonary hypoplasia. The mesothelium is a specialized epithelial tissue that lines the embryonic lung and has a major influence on lung growth by secreting growth factors such as FGF9 and serving as a progenitor pool for various cells in the growing lung. However, it is unclear what regulates these two phenomena. Previous data along with our preliminary data has led to the hypothesis that these functions of the mesothelium are regulated by tissue stretch, and this stretch is dysregulated during CDH. Herein, we propose the mechanism by which this stretch is sensed by the mesothelium, along with the downstream signaling that leads to eventual FGF9 expression. Overall, the mesothelium is understudied considering its major influence on lung growth, and we are the first to propose that the mesothelium is mechanically stimulated during development. In both Aims we will use our novel microfluidic chest cavity to examine the effects of mesothelial stretch on the whole tissue level. With this device, we can control the fluid pressure internal and external of a mouse lung explant to induce controlled stretch on the lung in a physiological 3D culture system. In our first Aim, we will test our proposed pathway involving stretch mediated FGF9 signaling. We hypothesize that this stretch is sensed via the mechanosensitive molecule YAP, which in other systems is stretch responsive. YAP then induces WT1 and RA signaling to cause FGF9 expression from the tissue. In addition to using our microfluidic chest cavity studying whole tissue effects, we will also apply controlled stretch to isolated primary mesothelial cells using microfluidic stretch membranes. We will test our hypothesis by examining the effect of stretch on the various molecules in the YAP/WT1/RA/FGF9 pathway, and the effect on growth when this pathway is interrupted. In our second Aim, we hypothesize that mesothelial stretch is sensed by YAP to induce epithelial-mesenchymal transition(EMT). The mesothelium has been shown to undergo EMT as serve as a progenitor pool for various lung tissue, and in other systems, YAP is a known inducer of EMT. We will determine the effect of stretch on mesothelial EMT.
As the lung is growing during development, a variety of forces act on the tissue. It is thought that disruption of these forces may contribute to the devastating condition Congenital Diaphragmatic Hernation (CDH) in infants. By understanding the specific effect of these forces on lung tissue and hence the influence on normal lung growth, we can understand how disruption of these forces relate to the cause of CDH and this information can guide development of improved treatment strategies to rescue lung growth in babies affected by this condition.
