Connecting molecular mechanisms and physical forces as regulators of tissue architecture during embryonic lung development

Abstract

Morphogenesis of the embryonic lung begins early in embryogenesis. By week 7 in humans and day 12 in mice, the lung begins a process known as branching morphogenesis. During this phase of lung development, the lung is rapidly growing and expanding the developing airways to establish the overall architecture of the lung. This process of branching morphogenesis is essential to create the final size and structures of the adult lung, and any interruptions in this process can have devastating consequences at and after birth. The early embryonic lung consists of three main tissue compartments: 1) the epithelium, 2) the mesenchyme, and 3) the mesothelium. It is well accepted that complicated molecular reciprocal signaling occurs between these three layers to control branching. In fact, most of what is currently understood of lung development is understood in the context of molecular signaling within the lung. However, a variety of evidence also supports a role for physical signals in controlling branching. Within the field of lung development, the role of these physical signals has not been a major focus of investigation and is therefore not very well understood. To better understand how physical forces integrate with molecular signaling during lung development, we investigated branching morphogenesis at three different length scales: 1) whole organ, 2) tissue scale, and 3) cellular scale. Our initial investigations began by interrogating physical forces that acted upon the entire lung. Using a Wt1-/- mouse model we observed gross lung abnormalities and branching defects present at the beginning of branching morphogenesis that became exacerbated at older gestations. When we quantified the gene expression differences in these lungs, we saw significantly decreased expression of key morphogenetic molecular signals. However, when we removed the Wt1-/- lungs from the native chest cavity environment and cultured them, the branching defects were recovered, yet the gene expression differences were not. This indicated that the gene expression differences were not responsible for the differences in branching observed. When we examined the lungs in situ using micro-CT, we observed a significant reduction in the space available within the chest cavity, and a decrease in space available for the lung to grow within. This altered chest cavity environment created a physical restraint on the lungs that resulted in morphological defects. These morphological defects were independent from the altered molecular signaling, demonstrating how physical forces can shape the overall architecture of the whole organ. We then investigated how two tissue layers of the lung physically interact, and how that relates to our current understanding of molecular signaling in the lung. The focus on molecular signaling as a driver of branching morphogenesis was largely due to classic experiments performed in the ‘60s and ‘70s. These experiments demonstrated that when distal mesenchyme from the lung was added to a separate host trachea, supernumerary branches would form along the trachea. When we repeated these experiments using a mouse expressing fluorescent airway smooth muscle (ASM), we determined that supernumerary branches only formed through gaps in the ASM. Despite the presence of soluble signals from the distal mesenchyme, wherever ASM was present, the airway epithelium was physically restricted from branching. Wherever the ASM was absent, airway branching could proceed. The physical presence of ASM dominated over other soluble morphogen signals present. As a stiffer tissue, ASM physically restricts the underlying epithelium and acts as a physical barrier to control branching morphogenesis and sculpt the lung architecture. This data presents a varied understanding of lung development, whereby molecular signals are not solely responsible for controlling branching morphogenesis, and ASM patterning can also regulate branching. Beyond the physical presence of ASM acting to control lung development, the ASM additionally plays a role in morphogenesis when it spontaneously contracts and moves lumenal fluid towards the distal regions of the developing airway. The local epithelium in these distal regions will deform and stretch in response to these contractions, and it is hypothesized that these deformations may control expression of key morphological signals. One candidate morphogen is thought to be vascular endothelial growth factor-a (Vegfa), that is secreted from the epithelium. VEGF is responsible for controlling vascular patterning and is observed as a strain sensitive gene in other tissues. To investigate whether VEGF was secreted in response to ASM contractions, we first quantified the strain within the epithelial cell layer that results after ASM contracts. We then developed a stretch device to apply controlled physiological strains to isolated primary epithelium. After straining these cells, we determined that lung epithelium expresses Vegfa in response to physiological strain. Because of the role of VEGF in controlling vascular patterning, the local deformations of lung epithelium during development may control morphogenesis of the pulmonary vascular system. This has important implications for creating an efficient lung structure where the lung epithelium and vasculature are in close proximity. This coordinated architecture is essential for proper gas exchange after birth. In summary, I demonstrate, at three difference length scales, how morphogenesis of the lung can be controlled by multiple physical forces, and how these forces are related to molecular signaling. These varied physical forces can control morphogenesis of tissue architectures during branching of the embryonic lung.