Doctoral Dissertation Defense – Rachel M. Gilbert

BME PhD Candidate, Rachel M. Gilbert, will be defending her dissertation.


Understanding How Physical Forces Regulate Tissue Architecture During Embryonic Lung Development

When:   Monday December 20 @ 2:30 PM EST
VIRTUAL:    Password: Lungs
Committee Chair:   Jason Gleghorn, PhD
Committee:  Dawn Elliott, PhD; Randy Duncan, PhD; Curtis Johnson, PhD


Morphogenesis of the embryonic lung begins early in embryogenesis. In humans this equates to approximately week 4, and in mice it begins at approximately embryonic day 9.5. 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 airway to establish the overall architecture of the lung. This process of branching morphogenesis is essential to creating the final size and structures of the adult lung, and any interruptions in this process can have devastating consequences after birth.

Most of what is currently understood of lung development is understood in the context of molecular signaling within the lung. 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. 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 the role of physical forces 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 defect 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 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 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 physical forces could act between two tissue layers of the lung to control morphogenesis. The focus on molecular signaling as a driver of branching morphogenesis was largely due to classic experiments. 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 noticed that in these graft experiments, supernumerary branches only formed through the gaps in the ASM along the trachea. Despite the presence of soluble signals from the distal mesenchyme, wherever ASM was present, the airway epithelium was physically restricted from branching, and wherever the ASM was absent, airway branching could proceed. The physical presence of ASM dominates over other soluble morphogen signals present. ASM exerts a physical force on the underlying epithelium, acting as a physical barrier to restrict branching morphogenesis and sculpt the lung architecture. Only in regions where ASM is absent can branching proceed.

The ASM additionally plays a role in morphogenesis when it spontaneously contraction and moves fluid towards the distal regions of the developing airway. The local epithelium will strain 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), which is responsible for controlling vascular patterning and known to be strain sensitive in other tissues. We first quantified the strain within the epithelial cell layer that results after ASM contracts. We then developed a mini stretch device to apply controlled physiological strains to isolated primary epithelium. After straining these cells, we determined that lung epithelium secretes 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 which is essential for proper gas exchange after birth.

In summary, we demonstrate at three difference length scales how morphogenesis of the lung can be controlled by multiple physical forces. These varied physical forces can control morphogenesis of tissue architectures during branching of the embryonic lung.